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.     

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.     

Proposal of Xanthomonas translucens pv. pistaciae pv. nov., pathogenic to pistachio (Pistacia vera)

Strains of Xanthomonas translucens have caused dieback in the Australian pistachio industry for the last 15 years. Such pathogenicity to a dicotyledonous woody host contrasts with that of other pathovars of X. translucens, which are characterized by their pathogenicity to monocotyledonous plant families. Further investigations, using DNA-DNA hybridization, gyrB gene sequencing and integron screening, were conducted to confirm the taxonomic status of the X. translucens pathogenic to pistachio. DNA-DNA hybridization provided a clear classification, at the species level, of the pistachio pathogen as a X. translucens. In the gyrB-based phylogeny, strains of the pistachio pathogen clustered among the X. translucens pathovars as two distinct lineages. Integron screening revealed that the cassette arrays of strains of the pistachio pathogen were different from those of other Xanthomonas species, and again distinguished two groups. Together with previously reported pathogenicity data, these results confirm that the pistachio pathogen is a new pathovar of X. translucens and allow hypotheses about its origin. The proposed name is Xanthomonas translucens pv. pistaciae pv. nov.     

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.     

Insights into the genome of the xanthan-producing phytopathogen Xanthomonas arboricola pv. pruni 109 by comparative genomic hybridization

The phytopathogenic bacterium Xanthomonas arboricola pv. pruni is the causal agent of Prunus Bacterial Spot disease that infects cultivated Prunus species and their hybrids. Furthermore, X. arboricola pv. pruni (Xap) plays a role in biotechnology since it produces xanthan gum, an important biopolymer used mainly in the food, oil, and cosmetics industry. To gain first insights into the genome composition of this pathovar, genomic DNA of X. arboricola pv. pruni strains was compared to the genomes of reference strains X. campestris pv. campestris B100 (Xcc B100) and X. campestris pv. vesicatoria 85-10 (Xcv 85-10) applying microarray-based comparative genomic hybridizations (CGH). The results implied that X. arboricola pv. pruni 109 lacks 6.67% and 5.21% of the genes present in the reference strains Xcc B100 and Xcv 85-10, respectively. Most of the missing genes were found to be organized in clusters and do not belong to the core genome of the two reference strains. Often they encode mobile genetic elements. Furthermore, the absence of gene clusters coding for the lipopolysaccharide (LPS) O-antigens of Xcc B100 and Xcv 85-10 indicates that the structure of the O-antigen of X. arboricola pv. pruni 109 differs from that of Xcc B100 and Xcv 85-10.     

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.     

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.     

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.     

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.     

New Multilocus Variable-Number Tandem-Repeat Analysis Tool for Surveillance and Local Epidemiology of Bacterial Leaf Blight and Bacterial Leaf Streak of Rice Caused by Xanthomonas oryzae

Multilocus variable-number tandem-repeat analysis (MLVA) is efficient for routine typing and for investigating the genetic structures of natural microbial populations. Two distinct pathovars of Xanthomonas oryzae can cause significant crop losses in tropical and temperate rice-growing countries. Bacterial leaf streak is caused by X. oryzae pv. oryzicola, and bacterial leaf blight is caused by X. oryzae pv. oryzae. For the latter, two genetic lineages have been described in the literature. We developed a universal MLVA typing tool both for the identification of the three X. oryzae genetic lineages and for epidemiological analyses. Sixteen candidate variable-number tandem-repeat (VNTR) loci were selected according to their presence and polymorphism in 10 draft or complete genome sequences of the three X. oryzae lineages and by VNTR sequencing of a subset of loci of interest in 20 strains per lineage. The MLVA-16 scheme was then applied to 338 strains of X. oryzae representing different pathovars and geographical locations. Linkage disequilibrium between MLVA loci was calculated by index association on different scales, and the 16 loci showed linear Mantel correlation with MLSA data on 56 X. oryzae strains, suggesting that they provide a good phylogenetic signal. Furthermore, analyses of sets of strains for different lineages indicated the possibility of using the scheme for deeper epidemiological investigation on small spatial scales.     

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.     

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.     

Identification of a major QTL for Xanthomonas arboricola pv. pruni resistance in apricot

Xanthomonas arboricola pv. pruni causes bacterial spot of stone fruit resulting in severe yield losses in apricot production systems. Present on all continents, the pathogen is regulated in Europe as a quarantine organism. Host resistance is an important component of integrated pest management; however, little work has been done describing resistance against X. arboricola pv. pruni. In this study, an apricot population derived from the cross “Harostar” × “Rouge de Mauves” was used to construct two parental genetic maps and to perform a quantitative trait locus analysis of resistance to X. arboricola pv. pruni. A population of 101 F1 individuals was inoculated twice for two consecutive years in a quarantine greenhouse with a mixture of bacterial strains, and disease incidence and resistance index data were collected. A major QTL for disease incidence and resistance index accounting respectively for 53 % (LOD score of 15.43) and 46 % (LOD score of 12.26) of the phenotypic variation was identified at the same position on linkage group 5 of “Rouge de Mauves.” Microsatellite marker UDAp-452 co-segregated with the resistance, and two flanking microsatellites, namely BPPCT037 and BPPCT038A, were identified. When dividing the population according to the alleles of UDAp-452, the subgroup with unfavorable allele had a disease incidence of 32.6 % whereas the group with favorable allele had a disease incidence of 21 %, leading to a reduction of 35.6 % in disease incidence. This study is a first step towards the marker-assisted breeding of new apricot varieties with an increased tolerance to X. arboricola pv. pruni.     

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.     

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.     

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.     

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.