Usefulness of nutritional screening for the identification of Xanthomonas campesths DNA homology groups and pathovars

A nutritional screen of 143 carbon sources was done on 88 strains of xanthomonads from 39 different Xanthomonas campestris pathovars, X. albilineans, X.fragariae , and ' X. gardneri '. Six compounds, cellobiose, fructose, fumarate, glucose, L-malate and succinate supported growth of all strains except X. albilineans , whereas 92 substrates were not utilized by any strain. Substrate utilization patterns appeared sufficiently uniform among the various genomic groups within Xanthomonas to allow their differentiation. The most easily distinguished pathovars were X. cam . pv. oryzicola and X. cam. secalis of genomic groups 4 and 3, respectively, because they used few substrates. Genomic group 1 was the most difficult to distinguish because utilization patterns differed substantially among the pathovars that comprise the group. Substrate utilization was useful for distinguishing pathovars within genomic groups. For example, X. campestris pv. pelargonii of genomic group 5 was differentiated from X. cam. carotae, X. cam. taraxaci , and ' X. gardneri ' by growth on aconitate but not D-tartrate. Similarly, use of D-tartrate differentiated X. celebensis from X. cam. pv. juglandis within group 6. Sorbitol was utilized only by X. cam. pv. plantaginis of group 2 and arabitol was a useful substrate for identifying X. cam. pv. pisi and pv. eucalypti . Most patterns of carbon utilization were confirmed with Biolog tests but there were exceptions as was found with utilization of glycerol and D-arabitol. The Biolog test also revealed some differences in carbon utilization not detected by standard tests of carbon substrates. It is concluded that nutritional screening has promise for identifying genomic groups and various pathovars within the genus Xanthomonas .     

Deoxyribonucleic acid relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae

Twenty Xanthomonas campestris pathotype strains, three non-pathotype strains, and one strain of X. fragariae were studied by S1 DNA:DNA hybridization tests. The results of these tests do not support the retention of X. campestris as a single species. DNA reassociation values among many of the strains were low. Three clusters of closely related strains were observed, but nine strains did not cluster. Xanthomonas campestris pv. secalis was more closely related to X. fragariae than to any other X. campestris pathovar. Mapping the host family upon a three-dimensional genomic distance matrix of the xanthomonads suggested that strains attacking the same plant family usually show some relationship, but only a distant one. Thus, pathogenicity toward members of the same host family is not a measure of the genomic relationships of xanthomonads.     

Genetic polymorphism in Xanthomonas albilineans strains originating from 11 geographical locations, revealed by two DNA probes

Two DNA fragments from Xanthomonas albilineans were used as probes to study the molecular diversity among strains of this pathogen. Two serologically distinct groups, serovars I and II, could be differentiated by hybridization to the probes. These probes, designated 830 and 838, were cloned after subtractive DNA hybridization of common sequences of Xanthomonas campestris pv. vasculorum from a serovar I strain of X. albilineans. They did not hybridize to the DNA of several other xanthomonads or to sugarcane DNA under the conditions of hybridization used. Faint bands were observed upon hybridization of probe 830 with one strain of X. campestris pv. phaseoli. The same banding patterns were obtained with a strain of X. albilineans from Burkina Faso and the serovar II strains of Mauritius. The serovar I strains from Mauritius and two other strains each from Reunion and South Africa had similar pattern.     

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.     

Differential expression of conserved protease genes in crucifer-attacking pathovars of Xanthomonas campestris.

Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.     

Differentiation between Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. oryzicola and the bacterial 'brown blotch' pathogen on rice by numerical analysis of phenotypic features and protein gel electrophoregrams

Thirty-five Xanthomonas campestris pv. oryzae, fourteen X. campestris pv. oryzicola strains and six 'brown blotch' pathogens of rice, all of different geographical origin, were studied by numerical analysis of 133 phenotype features and gel electrophoregrams of soluble proteins, %G + C determinations and DNA:rRNA hybridizations. The following conclusions were drawn. (i) The Xanthomonas campestris pathovars oryzae and oryzicola display clearly distinct protein patterns on polyacrylamide gels and can be differentiated from each other by four phenotype tests. (ii) Both pathovars are indeed members of Xanthomonas which belongs to a separate rRNA branch of the second rRNA superfamily together with the rRNA branches of Pseudomonas fluorescens, Marinomonas, Azotobacter, Azomonas and Frateuria. (iii) 'Brown blotch' strains are considerably different from X. campestris pv. oryzae and oryzicola. They are not members of the genus Xanthomonas, but are more related to the generically misnamed. Flavobacterium capsulatum, Pseudomonas paucimobilis, Flavobacterium devorans and 'Pseudomonas azotocolligans' belonging in the fourth rRNA superfamily. (iv) No correlation was found between the virulence, pathogenic groups or geographical distribution of X. campestris pv. oryzae or oryzicola strains and any phenotypic or protein electrophoretic property or clustering.     

Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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,     

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.     

A gene involved in quinate metabolism is specific to one DNA homology group of Xanthomonas campestris

A gene involved in quinate metabolism was cloned from Xanthomonas campestris pv. juglandis strain C5. The gene, qumA, located on a 4. 2-kb KpnI-EcoRV fragment in plasmid pQM38, conferred quinate metabolic activity to X. c. pv. celebensis. Tn3-spice insertional analyses further located the qumA gene on a region of about 3.0 kb within pQM38. Nucleotide sequencing of this 3.0-kb fragment reveals that the coding region of qumA is 2373 bp, the deduced amino acid sequence of which closely resembles a pyrrolo-quinoline quinone-dependent quinate dehydrogenase of Acinetobacter calcoaceticus. A 0.7 kb SalI-PstI fragment internal to qumA was used as a probe to hybridize against total genomic DNA from 43 pathovars of X. campestris. The fragment hybridized only to total genomic DNA from the four pathovars of DNA homology group 6, X. c. pv. celebensis, X. c. pv. corylina, X. c. pv. juglandis and X. c. pv. pruni, and from X. c. pv. carotae, which belongs to DNA homology group 5. This 0.7 kb fragment was also used as a probe to hybridize BamHI-digested total genomic DNAs from the four pathovars of DNA homology group 6 and X. c. pv. carotae. The restriction fragment length polymorphism pattern of DNA homology group 6 was different from that of X. c. pv. carotae. The probe hybridized to a 5.7-kb BamHI fragment in all four pathovars of group 6 and to a 6.1-kb BamHI fragment in three of four pathovars. It hybridized only to a 9. 9-kb BamHI fragment in X. c. pv. carotae. Quinate metabolism has previously been reported as a phenotypic property specific to X. campestris DNA homology group 6. Accordingly, a combination of the quinate metabolism phenotypic test and Southern hybridization using a qumA-derived probe will be very useful in the identification of pathovars in DNA homology group 6.     

Detection of Xanthomonas arboricola pv. pruni by PCR using primers based on DNA sequences related to the hrp genes

Efficient control of Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot on stone fruit, requires a sensitive and reliable diagnostic tool. A PCR detection method that utilizes primers to target the hrp gene cluster region was developed in this study. The nucleotide sequence of the PCR product amplified with primers specific for the hrp region of the xanthomonads and genomic DNA of X. arboricola pv. pruni was determined, and the sequence was aligned with that of X. campestris pv. campestris, which was obtained from the GenBank database. On the basis of the sequence of the amplified hrp region, a PCR primer set of XapF/R specific to X. arboricola pv. pruni was designed. This primer set yielded a 243-bp product from the genomic DNA of X. aboricola pv. pruni strains, but no products from other 21 strains of Xanthomonas or from two epiphytic bacterial species. Southern blot hybridization with the probe derived from the PCR product with the primer set and X. aboricola pv. pruni DNA confirmed the PCR results. The Xap primer system was successfully applied to detect the pathogen from infected peach fruits. When it was applied in field samples, the primer set was proved as a reliable diagnostic tool for specific detection of X. aboricola pv. pruni from peach orchards.     

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

Strains 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.     

Genetic Diversity of Xanthomonas arboricola pv. fragariae Strains and Comparison with some other X. arboricola Pathovars using Repetitive PCR Genomic Fingerprinting

The genetic relationship within 26 Xanthomonas arboricola pv. fragariae strains and between this pathovar and 20 strains of X. arboricola pv. corylina, 22 strains of X. arboricola pv. juglandis and 16 strains of X. arboricola pv. pruni has been assessed by means of repetitive polymerase chain reaction (rep‐PCR) using Enterobacterial Repetitive Intergenic Consensus), BOX (BOXA subunit of the BOX element of Streptococcus pneumoniae) and repetitive extragenic palindromic primer sets. Cluster analysis was performed by means of unweighted paired group method using arithmetic average (UPGMA). Upon rep‐PCR and UPGMA cluster analysis, a relevant genetic diversity was found within the strains. The overall similarity, however, was high (i.e. 80%). The four X. arboricola pathovars showed similar but clearly different genomic patterns and clustered into four different groups, with X. arboricola pv. corylina and X. arboricola pv. juglandis more closely related to X. arboricola pv. fragariae. Representative strains of X. arboricola pv. fragariae and the putative xanthomonads isolated from strawberry leaves showing leaf blight symptoms underwent pathogenicity tests. After artificial inoculation, X. arboricola pv. fragariae induced necrotic spots accompanied, sometimes, by a chlorotic halo. The blackening of the leaf veins and peduncle was, sometimes, also observed. The four putative xanthomonads isolated from diseased strawberry leaves and not inducing symptoms after artificial inoculation, clustered apart from X. arboricola pathovars.     

The A protein of the filamentous bacteriophage Cf of Xanthomonas campestris pv. citri.

Filamentous bacteriophages have very strict host specificities. Experiments were performed to investigate whether the A protein of the filamentous phage Cf, which infects Xanthomonas campestris pv. citri but not X. campestris pv. oryzae, is involved in determining Cf's host specificity. The gene encoding the A protein of Cf was cloned and expressed in X. campestris pv. citri. The genomic DNA of another filamentous bacteriophage, Xf, which infects X. campestris pv. oryzae but not X. campestris pv. citri, was then introduced by electroporation into X. campestris pv. citri that had expressed the A protein of Cf. The progeny phages thus produced were able to infect both X. campestris pv. oryzae and X. campestris pv. citri, indicating that the A protein of Cf was incorporated into the viral particles of Xf and conferred upon Xf the ability to infect the host of Cf. Inactivation of the A protein gene abolished the infectivity of Cf. The results of this study indicate that the A protein of Cf is responsible for controlling the host specificity of Cf.     

Identification of new candidate pathogenicity factors in the xylem-invading pathogen Xanthomonas albilineans by transposon mutagenesis

Xanthomonas albilineans is a xylem-invading pathogen that produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms of sugarcane leaf scald. In contrast to other xanthomonads, X. albilineans does not possess a hypersensitive response and pathogenicity type III secretion system and does not produce xanthan gum. Albicidin is the only previously known pathogenicity factor in X. albilineans, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. To identify additional host adaptation or pathogenicity factors, sugarcane 'CP80-1743' was inoculated with 1,216 independently derived Tn5 insertions in X. albilineans XaFL07-1 from Florida. Sixty-one Tn5 mutants were affected in development of leaf symptoms or in stalk colonization. The Tn5 insertion sites of these mutants were determined and the interrupted genes were identified using the recently available genomic DNA sequence of X. albilineans GPE PC73 from Guadeloupe. Several pathogenicity-related loci that were not previously reported in Xanthomonas spp. were identified, including loci encoding hypothetical proteins, a membrane fusion protein conferring resistance to novobiocin, transport proteins, TonB-dependent outer-membrane transporters, and an OmpA family outer-membrane protein.     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

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.