Characterization of ISXax1, a Novel Insertion Sequence Restricted to Xanthomonas axonopodis pv. phaseoli (Variants fuscans and non-fuscans) and Xanthomonas axonopodis pv. vesicatoria

ISXax1 is a novel insertion sequence belonging to the IS256 and Mutator families. Dot blot, Southern blot, and PCR analyses revealed that ISXax1 is restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and X. axonopodis pv. vesicatoria strains. Directed AFLP also showed that a high degree of polymorphism is associated with ISXax1 insertion in these strains.     

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.     

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.     

Geographical Differentiation of the Population of Xanthomonas axonopodis pv. manihotis in Colombia

Analyses of DNA polymorphism and virulence variation were used to evaluate the population structure of Xanthomonas axonopodis pv. manihotis, the pathogen causing cassava bacterial blight in Colombia. We collected strains from the major cassava-growing regions which can be grouped into different edaphoclimatic zones (ECZs) according to environmental conditions, production constraints, and economic parameters. DNA polymorphism was assessed by a restriction fragment length polymorphism analysis, using an X. axonopodis pv. manihotis plasmid DNA sequence (pthB) as a probe to evaluate the genetic relatedness among 189 Colombian strains. The sampling intensity permitted the estimation of genetic differentiation within and among ECZs, sites, and fields and even within an individual plant. A multiple correspondence analysis indicated that the Colombian X. axonopodis pv. manihotis population showed a high degree of diversity relative to X. axonopodis pv. manihotis populations studied previously, and the entire collection was grouped into seven clusters. A general correlation was observed between the clusters and the geographical origin of the strains, as each cluster was largely composed of strains from the same ECZ. Representative strains, identified with pthB, were further characterized by ribotyping, hybridization to two repetitive genomic probes (pBS6 and pBS8), and restriction analysis of plasmid contents to evaluate the complementarity of these markers. Virulence variation was observed within the Colombian collection. Strains of different aggressiveness were found in all ecological zones, but no correlation between virulence variation and DNA polymorphism was observed. The genetic and virulence analyses contribute to understanding the X. axonopodis pv. manihotis population structure in Colombia.     

Genetic structure and population dynamics of Xanthomonas axonopodis pv. manihotis in Colombia from 1995 to 1999

Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different X. axonopodis pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the X. axonopodis pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of X. axonopodis pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of X. axonopodis pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.     

Avirulence gene diversity of Xanthomonas axonopodis pv. glycines isolated in Korea

The hybridization patterns with the avrBs3 gene that is known to determine the recognition of host specificity were used to study the diversity of Xanthomonas axonopodis pv. glycines causing bacterial leaf pustule in soybean. A total of 155 strains were isolated from diverse tissues of soybean cultivars collected in Korea and were classified into six different type strains of OcsF, SL1017, SL1018, SL1045, SL1157, and SL2098 according to the patterns of avrBs3-homologous bands. When these type strains were inoculated on various cultivars, most of the Korean strains mildly induced disease symptoms on the resistant CNS1 cultivars. Unlike other type strains, strain SL2098, which appeared not to contain any avrBs3 homolog, induced only a few pustules on even highly susceptible cultivars. When a plasmid carrying the 3.7-kb avrBs3-homologous gene from strain SL1045 was introduced into SL2098, the transformant could not recover the pathogenicity in susceptible host plants. However, when avrBs3-homologous genes of strain SL1018 were mutated by transposon mutagenesis, one of the mutants in which a 5.2-kb chromosomal band homologous to avrBs3 was disrupted could not induce the hypersensitive response on resistant cultivars such as William82 or CNS2. Our results suggest that the avrBs3 homologs may play important roles in the pathogenicity of Xanthomonas axonopodis pv. glycines and the recognition of soybean cultivars.     

Mutation in the xpsD gene of Xanthomonas axonopodis pv. citri affects cellulose degradation and virulence

The Gram-negative bacterium Xanthomonas axonopodis pv. citri, the causal agent of citrus canker, is a major threat to the citrus industry worldwide. Although this is a leaf spot pathogen, it bears genes highly related to degradation of plant cell walls, which are typically found in plant pathogens that cause symptoms of tissue maceration. Little is known on Xac capacity to cause disease and hydrolyze cellulose. We investigated the contribution of various open reading frames on degradation of a cellulose compound by means of a global mutational assay to selectively screen for a defect in carboxymethyl cellulase (CMCase) secretion in X. axonopodis pv. citri. Screening on CMC agar revealed one mutant clone defective in extracellular glycanase activity, out of nearly 3,000 clones. The insertion was located in the xpsD gene, a component of the type II secretion system (T2SS) showing an influence in the ability of Xac to colonize tissues and hydrolyze cellulose. In summary, these data show for the first time, that X. axonopodis pv. citri is capable of hydrolyzing cellulose in a T2SS-dependent process. Furthermore, it was demonstrated that the ability to degrade cellulose contributes to the infection process as a whole.     

HrpG and HrpX play global roles in coordinating different virulence traits of Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri is the causal agent of citrus canker, which is one of the most serious diseases of citrus. To understand the virulence mechanisms of X. axonopodis pv. citri, we designed and conducted genome-wide microarray analyses to characterize the HrpG and HrpX regulons, which are critical for the pathogenicity of X. axonopodis pv. citri. Our analyses revealed that 232 and 181 genes belonged to the HrpG and HrpX regulons, respectively. In total, 123 genes were overlapped in the two regulons at any of the three selected timepoints representing three growth stages of X. axonopodis pv. citri in XVM2 medium. Our results showed that HrpG and HrpX regulated all 24 type III secretion system genes, 23 type III secretion system effector genes, and 29 type II secretion system substrate genes. Our data revealed that X. axonopodis pv. citri regulates multiple cellular activities responding to the host environment, such as amino acid biosynthesis; oxidative phosphorylation; pentose-phosphate pathway; transport of sugar, iron, and potassium; and phenolic catabolism, through HrpX and HrpG. We found that 124 and 90 unknown genes were controlled by HrpG and HrpX, respectively. Our results suggest that HrpG and HrpX interplay with a global signaling network and co-ordinate the expression of multiple virulence factors for modification and adaption of host environment during X. axonopodis pv. citri infection.     

Molecular and functional characterization of a unique sucrose hydrolase from Xanthomonas axonopodis pv. glycines

A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels. SUH was shown to act rather specifically on sucrose (K(m) = 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 +/- 12 Da, amino acid sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X. axonopodis pv. glycines 8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family 13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters.     

Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with novel markers and using a dot blot platform coupled with automatic data analysis

Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNA-based methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads.     

Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease, and related strains provides insights into virulence and host specificity

Xanthomonas axonopodis pv. citrumelo is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing, and optical mapping were used to obtain a complete genome sequence of X. axonopodis pv. citrumelo strain F1, 4.9 Mb in size. The strain lacks plasmids, in contrast to other citrus Xanthomonas pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen X. campestris pv. vesicatoria 85-10, with a completely different host range. We also compared X. axonopodis pv. citrumelo to the genome of citrus canker pathogen X. axonopodis pv. citri 306. Comparative genomic analysis showed differences in several gene clusters, like those for type III effectors, the type IV secretion system, lipopolysaccharide synthesis, and others. In addition to pthA, effectors such as xopE3, xopAI, and hrpW were absent from X. axonopodis pv. citrumelo while present in X. axonopodis pv. citri. These effectors might be responsible for survival and the low virulence of this pathogen on citrus compared to that of X. axonopodis pv. citri. We also identified unique effectors in X. axonopodis pv. citrumelo that may be related to the different host range as compared to that of X. axonopodis pv. citri. X. axonopodis pv. citrumelo also lacks various genes, such as syrE1, syrE2, and RTX toxin family genes, which were present in X. axonopodis pv. citri. These may be associated with the distinct virulences of X. axonopodis pv. citrumelo and X. axonopodis pv. citri. Comparison of the complete genome sequence of X. axonopodis pv. citrumelo to those of X. axonopodis pv. citri and X. campestris pv. vesicatoria provides valuable insights into the mechanism of bacterial virulence and host specificity.     

Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island

We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 micro M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

Reduced genetic variation occurs among genes of the highly clonal plant pathogen Xanthomonas axonopodis pv. vesicatoria, including the effector gene avrBs2

The bacterial plant pathogen Xanthomonas axonopodis pv. vesicatoria, also known as Xanthomonas campestris pv. vesicatoria group A, is the causal agent of bacterial spot in pepper and tomato. In order to test different models that may explain the coevolution of avrBs2 with its host plants, we sequenced avrBs2 and six chromosomal loci (total of 5.5 kb per strain) from a global sample of 55 X. axonopodis pv. vesicatoria strains collected from diseased peppers. We found an extreme lack of genetic variation among all X. axonopodis pv. vesicatoria genomic loci (average nucleotide diversity, pi = 9.1 x 10(-5)), including avrBs2. This lack of diversity is consistent with X. axonopodis pv. vesicatoria having undergone a recent population bottleneck and/or selective sweep followed by population expansion. Coalescent analysis determined that approximately 1.4 x 10(4) to 7.16 x 10(4) bacterial generations have passed since the most recent common ancestor (MRCA) of the current X. axonopodis pv. vesicatoria population. Assuming a range of 50 to 500 bacterial generations per year, only 28 to 1,432 years have passed since the MRCA. This time frame coincides with human intervention with the pathogen's host plants, from domestication to modern agricultural practices. Examination of 19 mutated (loss-of-function) avrBs2 alleles detected nine classes of mutations. All mutations affected protein coding, while no synonymous changes were found. The nature of at least one of the avrBs2 mutations suggests that it may be possible to observe one stage of an evolutionary arms race as X. axonopodis pv. vesicatoria responds to selection pressure to alter avrBs2 to escape host plant resistance.     

Genome sequence of Xanthomonas axonopodis pv. punicae strain LMG 859

We report the 4.94-Mb genome sequence of Xanthomonas axonopodis pv. punicae strain LMG 859, the causal agent of bacterial leaf blight disease in pomegranate. The draft genome will aid in comparative genomics, epidemiological studies, and quarantine of this devastating phytopathogen.     

A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.     

A pthA homolog from Xanthomonas axonopodis pv. citri responsible for host-specific suppression of virulence

Strains of the plant-pathogenic bacterium Xanthomonas axonopodis pv. citri are differentiated into two groups with respect to aggressiveness (normal and weak) on Citrus grandis cultivars but not on other Citrus species such as Citrus sinensis. Random mutagenesis using the transposon Tn5 in X. axonopodis pv. citri strain KC21, which showed weak aggressiveness on a C. grandis cultivar, was used to isolate mutant KC21T46, which regained a normal level of aggressiveness on the cultivar. The gene inactivated by the transposon, hssB3.0, was shown to be responsible for the suppression of virulence on C. grandis. Sequence analysis revealed it to be a new member of the pthA homologs, which was almost identical in sequence to the other homologs except for the number of tandem repeats in the central region of the gene. hssB3.0 appears to be a chimera of other pthA homologs, pB3.1 and pB3.7, and could have been generated by recombination between these two genes. Importantly, in X. axonopodis pv. citri, hssB3.0 was found in all of the tested isolates belonging to the weakly aggressive group but not in the isolates of the normally aggressive group. Isolation of the virulence-deficient mutant KC21T14 from KC21, in which the pathogenicity gene pthA-KC21 was disrupted, showed that hssB3.0 induces a defense response on the host but partially interrupts canker development elicited by the pathogenicity gene in this bacterium.