Deoxyribonucleic acid relatedness of 21 strains of Xanthomonas species and pathovars

The relationship of 17 Xanthomonas campestris pathotype strains, three additional X. campestris strains, and the type strain of Xanthomonas albilineans were examined by DNA-DNA hybridization tests. The results coupled with those of a previous study (Hildebrand et al. 1990) support the hypothesis that X. campestris does not constitute a single bacterial species. There were low levels of DNA-DNA reassociation among many of the different pathovars examined. Six clusters of related pathovars were discerned. In addition, four of the pathovars were only distantly related to each other and to the six clusters. Xanthomonas albilineans was not closely related to any of the other xanthomonads tested.
Mapping and superimposing the botanical families of the host plants upon a three-dimensional genomic distance matrix of the xanthomonads confirms previous observations that pathovars that infect plants of the same botanical family do not necessarily belong to the same genomic group. Six legume-infecting pathovars cluster within one genomic group, but one pathovar, X. cam. pv. pisi is only distantly related to this group. There was also no genomic relationship between X. cam. pv. oryzicola and X. albilineans both of which infect Gramineae. Consequently, pathogenicity toward members of the same plant family is not a good indicator of the genomic relationships among xanthomonads nor is it a good taxonomic determinant.     

Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10³ CFU ml⁻¹) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

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.     

Integron variability in Xanthomonas arboricola pv. juglandis and Xanthomonas arboricola pv. pruni strains

The integron platform and the gene cassette arrays of 34 Xanthomonas arboricola pv. juglandis and of 47 Xanthomonas arboricola pv. pruni strains isolated from different geographical areas were screened to check their variability. Genetic variability of the strains was also tested by means of BOX-PCR. For two representative strains of the two pathovars, the integrase gene intI and part of the flanking gene ilvD were also cloned and sequenced. Whereas X. a. pv. pruni strains did not show relevant variability, six X. a. pv. juglandis strains isolated in Australia showed some differences in the gene sequences. The CLUSTALW algorithm indicated that the majority of the X. a. pv. juglandis strains are closely related to X. a. pv. pruni, whereas the X. a. pv. juglandis strains isolated in Australia were more similar to Xanthomonas hortorum pv. pelargonii. Similarly, the gene cassette array pattern of the Australian strains, as well as that of the oldest strain maintained in culture, was different from the other strains. Also, three X. a. pv. pruni strains showed a different cassette array pattern when compared with the majority of other strains but no relationships with geographical area of isolation or host plant was revealed. This study confirmed that in addition to species, integrons may generate diversity also within two X. arboricola pathovars.     

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.     

Phylogenetic relatedness of Xanthomonas axonopodis pv. punicae, the causal agent of bacterial blight of pomegranate based on two loci, 16S rRNA and gyrB

Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease in pomegranate (Punica granatum) cultivation in India. The Xap strains from three distinct geographical origins, Delhi, Maharashtra and Andhra Pradesh were studied for their genetic variability and phylogenetic relationship with other Xanthomonads targeting two important loci 16S rRNA and gyrB. All Xap strains showed 100 % sequence conservation in both the loci, suggesting that geographical origin does not necessarily reflect variation to genetic make-up of the Xap. Phylogeny derived from 16S rRNA gene revealed that two Xanthomonas species, Xanthomonas citri subsp. malvacearum DSM 3849 T and X. axonopodis pv. manihotis NCPPB1834 formed a single cluster along with Xap. Further, analysis in the gyrB locus indicated that X. citri subsp. malvacearum shared 99.4 % identity while pathovars X. axonopodis pv. manihotis shared only 95 % identity with the Xap strains. Thus, we established that gyrB was the preferred locus over 16S rRNA gene to discriminate the Xap strains from closely related Xanthomonas species type strains. Nevertheless, our study demonstrated for the first time that pomegranate bacterial blight pathogen is phylogenetically very close to Xanthomonas citri subsp. malvacearum infecting cotton.     

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 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.     

Genetic Characterization of Pseudomonas syringae pv. syringae Strains from Stone Fruits in California

Strains of Pseudomonas syringae pv. syringae were isolated from healthy and diseased stone fruit tissues sampled from 43 orchard sites in California in 1995 and 1996. These strains, together with P. syringae strains from other hosts and pathovars, were tested for pathogenicity and the presence of the syrB and syrC genes and were genetically characterized by using enterobacterial repetitive intergenic consensus (ERIC) primers and PCR. All 89 strains of P. syringae pv. syringae tested were moderately to highly pathogenic on Lovell peach seedlings regardless of the host of origin, while strains of other pathovars exhibited low or no pathogenicity. The 19 strains of P. syringae pv. syringae examined by restriction fragment length polymorphism analysis contained the syrB and syrC genes, whereas no hybridization occurred with 4 strains of other P. syringae pathovars. The P. syringae pv. syringae strains from stone fruit, except for a strain from New Zealand, generated ERIC genomic fingerprints which shared four fragments of similar mobility. Of the P. syringae pv. syringae strains tested from other hosts, only strains from rose, kiwi, and pear generated genomic fingerprints that had the same four fragments as the stone fruit strains. Analysis of the ERIC fingerprints from P. syringae pv. syringae strains showed that the strains isolated from stone fruits formed a distinct cluster separate from most of the strains isolated from other hosts. These results provide evidence of host specialization within the diverse pathovar P. syringae pv. syringae.     

Rapid Identification of Xanthomonas campestris pv. graminis and X.c. pv. phlei by Fatty Acid Profiling

Xanthomonas campestris pv. graminis and X. campestris pv. phlei isolated from different grass-species were analysed for their fatty acid content with a gas-chromatograph and a commerially-available software package. The two pathovars could be rapidly and reliably identified and separated from each other with this technique, offering alternative to time-consuming identification by biochemical and pathogenicity tests.     

Genomic Analysis of Xanthomonas translucens Pathogenic on Wheat and Barley Reveals Cross-Kingdom Gene Transfer Events and Diverse Protein Delivery Systems

In comparison to dicot-infecting bacteria, only limited numbers of genome sequences are available for monocot-infecting and in particular cereal-infecting bacteria. Herein we report the characterisation and genome sequence of Xanthomonas translucens isolate DAR61454 pathogenic on wheat and barley. Based on phylogenetic analysis of the ATP synthase beta subunit (atpD) gene, DAR61454 is most closely related to other X. translucens strains and the sugarcane- and banana- infecting Xanthomonas strains, but shares a type III secretion system (T3SS) with X. translucens pv. graminis and more distantly related xanthomonads. Assays with an adenylate cyclase reporter protein demonstrate that DAR61454''s T3SS is functional in delivering proteins to wheat cells. X. translucens DAR61454 also encodes two type VI secretion systems with one most closely related to those found in some strains of the rice infecting strain X. oryzae pv. oryzae but not other xanthomonads. Comparative analysis of 18 different Xanthomonas isolates revealed 84 proteins unique to cereal (i.e. rice) infecting isolates and the wheat/barley infecting DAR61454. Genes encoding 60 of these proteins are found in gene clusters in the X. translucens DAR61454 genome, suggesting cereal-specific pathogenicity islands. However, none of the cereal pathogen specific proteins were homologous to known Xanthomonas spp. effectors. Comparative analysis outside of the bacterial kingdom revealed a nucleoside triphosphate pyrophosphohydrolase encoding gene in DAR61454 also present in other bacteria as well as a number of pathogenic Fusarium species, suggesting that this gene may have been transmitted horizontally from bacteria to the Fusarium lineage of pathogenic fungi. This example further highlights the importance of horizontal gene acquisition from bacteria in the evolution of fungi.     

Specific detection of Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andreanum) tissues by nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10(3) CFU ml(-1)) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.     

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 relationship between PthA expression and the pathogenicity of Xanthomonas axonopodis pv. citri

Citrus canker disease, caused by Xanthomonas axonopodis pv. citri, affects almost all citrus species and cultivars and hascaused severe damage to the citrus industry worldwide. PthA is considered the main pathogenesis effector of the pathogen. This research aimed to temporally and spatially analyze the expression of the PthA protein of the bactrium during its culture, and then try to understand the relationship between the PthA expression levels and the pathogenicity. The relationship between the expression of PthA and the pathogenicity of X. axonopodis pv. citri was fully investigated by using SDS-PAGE, Western blot, ELISA and field inoculation, It was found that bacteria cultured for 36 h had the highest expression of PthA and showed the most virulent pathogenicity. The conservation duration of the pathogen isolates influenced their PthA expression and the pathogenicity, and negative relationship between the duration and the expression of PthA and pathogenicity. When the stored pathogen bacteria were cultured in liquid LB medium, they were able to regain activated, showing higher PthA expression level and enhanced pathogenicity, even though the activity was inferior, in terms of both PthA expression and pathogenicity, than the freshly isolated ones. Seven isolates from different citrus orchards displayed almost identical protein expression profiles. It could conclude that the expressions of PthA was positively related to pathogenicity.     

Development of Host Range Mutants of Xanthomonas campestris pv. translucens

Xanthomonas campestris pv. translucens is the causal agent of bacterial leaf streak of cereal grains and grasses, and individual strains within the pathovar differ in their host range among the cereals. Coinoculation of a wide-host-range and a narrow-host-range strain resulted in the wide-host-range reaction. Transposon and chemical mutagenesis of the wide-host-range strain Xct4, pathogenic on barley, wheat, rye, and triticale, resulted in variants with reduced host range. When pathogenicity was inactivated independently for barley, wheat, triticale, and rye, wild-type symptoms were retained on the other members in the host range. Testing of some host range mutants on additional varieties of the cereals indicated some cultivar specificity. In addition, mutants nonpathogenic on combinations of the hosts or on all hosts were isolated. This suggests that there are independent positive factors determining host range in this species, rather than an avirulence gene system such as those determining race specificity in other plant pathogens.     

The ubiquitous plasmid pXap41 in the invasive phytopathogen Xanthomonas arboricola pv. pruni: complete sequence and comparative genomic analysis

The complete DNA sequence of the 41 102-bp plasmid pXap41 from the invasive plant pathogen Xanthomonas arboricola pv. pruni CFBP 5530 was determined and its 44 coding regions were annotated. Comparative analysis with 15 Xanthomonas plasmids and 19 complete genomes revealed that nearly one-fourth of this plasmid has high sequence identity to plasmid pXAC64 and an 8.8-kb chromosomal region of Xanthomonas axonopodis pv. citri strain 306 carrying genes that encode type III effectors and helper proteins. The presence of pXap41 in all X. arboricola pv. pruni genotypes was confirmed for eight strains by plasmid profiling and for 35 X. arboricola pv. pruni isolates with a new plasmid multiplex PCR assay. This plasmid was not detected in any other X. arboricola pathovars (n=12), indicating the potential for the application of the pXap41 PCR method as a pathovar-level detection and identification tool.     

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.     

Phylogenetic and Variable-Number Tandem-Repeat Analyses Identify Nonpathogenic Xanthomonas arboricola Lineages Lacking the Canonical Type III Secretion System

Xanthomonas arboricola is conventionally known as a taxon of plant-pathogenic bacteria that includes seven pathovars. This study showed that X. arboricola also encompasses nonpathogenic bacteria that cause no apparent disease symptoms on their hosts. The aim of this study was to assess the X. arboricola population structure associated with walnut, including nonpathogenic strains, in order to gain a better understanding of the role of nonpathogenic xanthomonads in walnut microbiota. A multilocus sequence analysis (MLSA) was performed on a collection of 100 X. arboricola strains, including 27 nonpathogenic strains isolated from walnut. Nonpathogenic strains grouped outside clusters defined by pathovars and formed separate genetic lineages. A multilocus variable-number tandem-repeat analysis (MLVA) conducted on a collection of X. arboricola strains isolated from walnut showed that nonpathogenic strains clustered separately from clonal complexes containing Xanthomonas arboricola pv. juglandis strains. Some nonpathogenic strains of X. arboricola did not contain the canonical type III secretion system (T3SS) and harbored only one to three type III effector (T3E) genes. In the nonpathogenic strains CFBP 7640 and CFBP 7653, neither T3SS genes nor any of the analyzed T3E genes were detected. This finding raises a question about the origin of nonpathogenic strains and the evolution of plant pathogenicity in X. arboricola. T3E genes that were not detected in any nonpathogenic isolates studied represent excellent candidates to be those responsible for pathogenicity in X. arboricola.