Xanthomonas Campestris Pathovar Research Articles

A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule.

Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris (Xcc). The phenotype of mutants in one of the genes, rpfF, can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipoplysaccharide and lipid A from Xcc, and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.

Variation for Black Rot Resistance in Tronchuda Regenerated from Epicotyl Segments

Tronchuda (Brassica oleracea var. tronchuda Bailey syn. costata) regenerants with resistance to Xanthomonas campestris pathovar campestris Pammel (Dawson) were produced by culturing epicotyl segments of 3-week-old seedlings on Murashige and Skoog medium supplemented with 2 mg BAP and NAA at 0.1 mg·liter–1. Cultures were placed in darkness (1, 2, or 3 weeks) or in a 16-hour·day–1 light regime. Seedlings of all four cultivars were more susceptible (mean disease severity rating of 3.8 to 4.0; where 1 = trace and 4 = more than 2 cm2 diseased leaf tissue) than the respective regenerants. There were significant differences in disease severity of regenerants among and within the four cultivars. Dark incubation of cultured explants generated plants with higher disease resistance than evident with control plants. One week of dark incubation resulted in increased resistance in regenerants of `Penca de Chaves', `Portuguesa', and `Vilinda' (2.1, 2.3, and 2.7 mean disease severity, respectively), whereas 2 weeks of dark incubation increased resistance in regenerants of `Ana Maria' and `Vilinda' (2.1 and 2.7 mean disease severity, respectively). The genotype×plant treatment interaction was significant. Chemical names used: 6-benzylaminopurine (BAP); a-naphthaleneacetic acid (NAA).

Electrotransformation of three pathovars of Xanthomonas campestris

Procedures for electrotransformation have been adapted for three pathovars of Xanthomonas campestris: campestris, vesicatoria and manihotis. Three differently sized plasmids (51, 9.3 and 3.3 kb) at different concentrations (10, 30 and 50 ng/sample) and different field strengths (10, 12, 14 and 18 kV/cm) were used. The efficiency of transformation was dependent on the recipient strain and the plasmid introduced. In general, a field strength of 14 kV/cm as well as a concentration of 30 ng plasmid DNA/sample seemed to be adequate for most conditions. Only X. campestris pv. vesicatoria strain 479 required a higher field strength for better efficiency. The plasmid size was inversely related to the efficiency of transformation; up to 1.6 × 1010, 5.3 × 107 and 3.7 × 105 transformants/μg DNA were obtained using the pXG31 (3.3 kb), pUFR027 (9.3 kb) and RP4 (51 kb) plasmids, respectively.

Testing for Pseudomonas syringae pv. atrofaciens and Xanthomonas campestris pathovars on cereals in Italy

During recent years, recurrent attacks of basal glume rot/leaf blight attributed to Pseudomonas syringae pv. atrofaciens have been observed on cultivars of durum wheat grown in the Po valley (IT). The main aim of this study was to test for this pathogen and Xanthomonas campestris pathovars in commercial seed lots of cereals produced in different regions of Italy, as well as in symptomatic plants collected in the field. None of the analyses led to the detection of xanthomonads. In testing for P.s. atrofaciens, representative bacterial isolates were selected and characterized; this was done by combining conventional identification tests and computerized densitometric analysis of electrophoretic patterns of cell proteins obtained using the SDS‐PAGE technique. P.s. atrofaciens was detected in seed samples (barley, wheat and durum wheat) and symptomatic plants (durum wheat) grown in northern and central Italy. The possible pathogenic role of other unrelated fluorescent pseudomonads in this interaction was also examined. Difficulties in discriminating pvs syringae and atrofaciens of P. syringae are discussed and the uncertain taxonomic relationship between these two pathovars is emphasized.

Bacterial leaf streak or black chaff of cereals

The literature related to bacterial leaf streak of cereals, due to various pathovars of Xanthomonas campestris, is confusing and dispersed. This review presents a synthesis of the knowledge that has been accumulated on the disease in cereals since the early reports on barley and wheat. The aim is to present the current status on geographical distribution of the disease, to explain possible confusions in symptoms, and to show the limited quantitative information available on losses and on host/pathogen interactions. Disease etiology and means of control are also reviewed. The objective is to define the present situation of black chaff in wheat, barley and triticale, and to delineate the major research needs in these crops. Difficulties in controlling bacterial leaf streak arise as a result of limited epidemiological knowledge and of the absence of satisfactory seed treatment permitting the eradication of the bacterium. There is an urgent need for a seed certification scheme and for more information on genetic resistance, with a view to limiting outbreaks of this sporadic but devastating disease.

Amplified DNA Polymorphisms of Putative Two-Component Regulatory Genes of Several Xanthomonas Campestris Pathovars

Amplified DNA Polymorphisms of Putative Two-Component Regulatory Genes of Several Xanthomonas Campestris Pathovars

Defense-related gene induction in Brassica campestris in response to defined mutants of Xanthomonas campestris with altered pathogenicity.

We have studied the induction of beta-1,3-glucanase (BGL) in turnip following inoculation with pathovars of Xanthomonas campestris and derived mutants. BGL transcript accumulated more rapidly in leaves in the incompatible interactions with X. c. pv. armoraciae and X. c. pv. raphani than in the compatible interaction with X. c. pv. campestris. No accumulation was seen in response to wounding or inoculation with water, salicylic acid, or Escherichia coli. Deletion of the hrp cluster from the X. campestris pathovars caused a reduction in the level of transcript accumulation; these effects were much more pronounced in the incompatible than in the compatible interaction, in which bacterial growth was also affected. In the compatible interaction, bacterial growth and BGL transcript accumulation were not altered by mutation of bacterial genes involved in the regulation of the synthesis of extracellular enzymes or their export from the cell, or by mutation of the structural genes for extracellular endoglucanase and serine protease. Mutation of genes involved in the synthesis of extracellular polysaccharide or lipopolysaccharide reduced bacterial survival in planta, so that the numbers were between two and three orders of magnitude lower than the number of wild-type bacteria. However, total BGL transcript accumulation after inoculation with these mutants was about 80% of that seen after inoculation with the wild-type bacteria, suggesting that one aspect of the role of extracellular polysaccharide and lipopolysaccharide in pathogenesis is to mask the presence of bacteria in the plant. Our results are discussed in the context of work on other plant-microbe interactions.

Pathogenicity determinants and global regulation of pathogenicity of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pathovar campestris (hereafter X.c. campestris), the causal agent of black rot of crucifers, is a phytopathogenic bacterium of great economic importance (WILLIAMS 1980; ONSANDO 1992; SCHAAD and ALVAREZ 1993). Molecular genetic methods have now allowed a number of genes encoding pathogenicity determinants of X.c. campestris and related pathovars to be identified. In this chapter we will briefly review some of these and discuss our current understanding of the regulation of pathogenicity in this bacterium.

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.

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.

Application of Fatty Acid Methyl Esters for the Taxonomic Analysis of the Genus Xanthomonas

Summary Gas-liquid chromatographic analysis was applied to examine whole-cell fatty acid methyl esters (FAMEs) of a total of 975 strains which included representatives of all seven Xanthomonas species ( X. albilineans, X. axonopodis, X. campestris, X. fragariae, X. maltophilia, X. oryzae and X. populi ), 134 X. campestris pathovars and two X. oryzae pathovars as well as some related strains. At least 65 different fatty acids were found within the members of the genus Xanthomonas . A set of nine fatty acids (11:0 iso, 11:0 iso 3OH, 12:0 3OH, 13:0 iso 3OH, 15:0 iso, 16:1 cis 9, 16:0, 17:1 iso F and 17:0 iso) occurred in more than 99% of the 966 genuine Xanthomonas strains and are therefore considered as the most common fatty acids for the genus Xanthomonas . The three fatty acids 11:0 iso, 11:0 iso 3OH and 13:0 iso 3OH are characteristic for all members of the genus Xanthomonas and serve as a useful criterion to differentiate Xanthomonas from other bacteria. Cluster analysis revealed 31 major FAME clusters within the genus Xanthomonas . The Xanthomonas species X. albilineans, X. axonopodis, X. fragariae, X. maltophilia and X. populi each constituted a separate FAME cluster. Two FAME clusters were formed within X. oryzae , corresponding to pvs. oryzae and oryzicola , respectively. The species X. campestris was demonstrated to be heterogeneous and comprised 24 FAME clusters. In some cases, X. campestris pathovars which were isolated from related host plants grouped together. This was the case for the following pathovars from grasses: X. c. pvs. graminis, poae and phleipratensis (FAME cluster 7); X. c. pvs. arrhenatheri and phlei (FAME cluster 8), and also for the following X. campestris pathovars from cereals: X. c. pvs. cerealis, hordei, undulosa, secalis and translucens (FAME cluster 9). All six X. campestris pathovars from the crucifers ( X. c. pvs. aberrans, armoraciae, barbareae, campestris, incanae and raphani ) fell within FAME cluster 2. Many Xanthomonas campestris pathovars from legumes were grouped within FAME cluster 1. Although most X. campestris pathovars were homogeneous or had only one or a few atypical strains, some X. campestris pathovars were found to be heterogeneous. The greatest heterogeneity occurred in X. c. pvs. vasculorum and citri , forming 4 and 3 FAME subgroups, respectively. Other X. campestris pathovars which constituted more than one FAME subgroup included X. c. pvs. phaseoli and vignicola . The comparison of the results of FAME analysis and those of phenotypic tests, SDS-PAGE of whole-cell proteins and DNA-DNA hybridization is discussed. A database generated by the authors based on the fatty acid compositions of Xanthomonas allows rapid identification of unknown xanthomonads at the genus, species and often at pathovar level.

Direct and indirect conductimetry for identification and detection of plant pathogenic bacteria

An automated conductimetric system was used to study the responses of some plant pathogenic bacteria in direct and indirect conductimetry. Strains of pathovars of Pseudomonas syringae and Xanthomonas campestris, one strains of Clavibacter michiganensis ssp. michiganensis Erwinia carovora ssp. atroseptica and Erwinia chrysanthemi were tested in Special Peptone Yeast Extract (SPYE), Malthus Columbia Broth (MCB) and Malthus Enterobacteriaceae Medium (MEM). In direct conductimetry the erwinias gave significantly lower detection times (the detection time was defined in direct conductimetry as the time to reach a conductance change of 100 μS) and much higher rates of conductance changes in SPYE and MCB than the pseudomonads and xanthomonads tested at 27 o C

Plant chitinases

Plant chitinases

Evaluation of a commercial microbial identification system based on fatty acid profiles for rapid, accurate identification of plant pathogenic bacteria

D.E. STEAD, J.E. SELLWOOD, J. WILSON AND I. VINEY, 1992. Fatty acid profiles of 773 strains representing 25 taxa of plant pathogenic and related saprophytic bacteria were compared with two commercially available broad‐spectrum libraries and one self‐generated library based primarily on cultures from the National Collection of Plant Pathogenic Bacteria. The accuracy of identification at specific level was often 100%, although for some closely related species and infraspecific taxa accuracy was sometimes significantly less than this. The accuracy of identification of Xanthomonas campestris pathovars was much better than for Pseudomonas syringae pathovars. Almost all identifications were made within24–48 h. Standardization of cultural conditions was essential. Hydroxy fatty acids were of great taxonomic value in classification of Gram‐negative bacteria. Improved library development and standardization of cultural and analytical techniques will further increase the accuracy of identification. Fatty acid profiling offers a valuable rapid, accurate method for identification of many bacteria.

Race-specificity of plant resistance to bacterial spot disease determined by repetitive motifs in a bacterial avirulence protein

ELUCIDATION of the genetic and molecular basis of plant disease resistance is a major objective in the investigation of plant-micro-bial interactions. Xanthomonas campestris pathovar vesicatoria (Xcv), the causal agent of bacterial spot disease of pepper and tomato, has been developed as a model host–pathogen system to study the genetic interactions that specify the expression of plant disease resistance1–6. Several plant resistance genes (Bsl, Bs2, Bs3) have been genetically characterized from pepper (Capsicum annuum) that determine resistance to particular races of the pathogen carrying specific avirulence genes7–9. For example, pepper plants carrying the resistance locus Bs3 are resistant to Xcv strains expressing the avirulence gene avrBs3. Nucleotide sequence analysis of the avrBs3 gene revealed that the internal portion of the predicted protein product consists of a nearly identical 34 amino acid repeat unit, present in 17.5 copies4. We report here that the repetitive region of the avrBs3 gene determines race-specificity and that deletions of repeat units generate new avirulence specificities and unmask undiscovered resistance genes in pepper and tomato.

Use of Quinate Metabolism as a Phenotypic Property to Identify Members ofXanthomonas campestrisDNA Homology Group 6

Certain pathovars of Xanthomonas campestris that metabolize quinate were discovered. This discovery led to the development of a succinate-quinate (SQ) medium for the detection of quinate metabolism. Of five species of Xanthomonas and 77 pathovars of X. campestris, only members of five pathovars of X. campestris were positive in the SQ test. All tested strains of the four pathovars of DNA homology group 6 (i.e., X. c. celebensis, X. c. corylina, X. c, juglandis, and X. c. pruni) were positive, except for two strains of X, c. celebensis (...)

A Device for Precise and Nondisruptive Stomatal Inoculation of Leaf Tissue with Bacterial Pathogens

A stomatal inoculation apparatus (SIA) was developed to produce water congestion of leaf tissues and provide a reproducible noninjurious means of introducing two Xanthomonas campestris pathovars of citrus into leaf tissues without wounding. The SIA consisted of a small inoculation chamber attached to an intact leaf. Water and inoculum were metered into an airstream and focused to impact on a 1-mm-diameter area of the leaf surface. Leaf tissues on the abaxial surface of Duncan grapefruit leaves expanded 50-75% were more susceptible to infection than were other growth stages (...)

Comparison of Immunofluorescence and Two Assays for Detection ofXanthomonas campestrispv.undulosain Seeds of Small Grains

A monoclonal antibody specific for Xanthomonas campestris pathovars in the translucens group, including X. c. pv. undulosa, the causal agent of bacterial leaf streak or black chaff in wheat and triticale, was used in immunofluorescence and a dot-immunobinding assay to detect pathogens in aqueous seed extracts. These techniques were compared to dilution plating of seed wash water as potential routine seed-indexing methods. Twenty-six cereal seed lots were harvested from plants with different levels of natural disease incidence grown in three locations in Mexico. Among the 26 lots analyzed, pathogen-free seed came from clean fields principally in Ciudad Obregon, where conditions are less favorable for its development. Pathogen populations detected with dilution plate ranged from 1.3 X 10(4) to 5.3 X 10(6) cfu/g of air-dry weight. Populations were recovered from seed lots that had been stored for 3 yr. Some discrepancies in pathogen detection occurred among the different methods used. Seed lots with high pathogen populations were consistently identified with all three methods. Differences in detection thresholds were found with seed wash water as compared to reference strain from pure culture. Apparently a substantial number of dead cells were present in wash water from some seed lots. Immunofluorescence was more sensitive than dot-immunobinding assay and produced more reproducible results. It is proposed as a standard for indexing germ plasm. Because of the high variation in data, direct counts of fluorescent bacterial cells under UV microscopy will probably not be necessary for identifying infected seed. It is recommended that immunofluorescence-positive seed lots should not be considered for sowing in areas favorable for disease development.

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

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

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with Mr values of 58,854, 42,299, 15,548, 18,214 and 15,108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 bp duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the PulF and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.