Klaus Rudolph

Levan and levansucrase synthesized by the fireblight pathogen Erwinia amylovora

The fireblight pathogen Erwinia amylovora synthesizes two different types of extracellular polysaccharide (EPS) when cultured in vitro, an acidic and a neutral compound. Neutral EPS was prepared from culture supernatants by cross-flow ultrafiltration and molecular washing, and purified by column chromatography. This material represented most of the crude EPS, which was produced in high-sucrose medium. It was entirely composed of fructose. The signals obtained from the EPS-preparation by 13C-NMR spectroscopy were identical with those of levan from Aerobacter levanicum indicating a β-2,6 linkage of the sugar residues. The neutral component of the EPS was thus identified as levan (β-2,6-d-fructofuranan). In the protein fraction associated with crude EPS, the levan polymerizing enzyme levansucrase (β-2,6-d-fructan: d-glucose 6-fructosyltransferase, EC 2.4.1.10) was detected. The enzyme was also produced at similar levels with several carbon sources by the bacterium. In polyacrylamide gel eletrophoresis its molecular weight under completely denaturating conditions was 46 kDa, when purified to apparent homogeneity by membrane-exchange chromatography. However, in isoelectric focussing, the enzyme preparation produced two bands at pI 4·0 and pI 3·6, respectively. Both bands displayed enzyme activity in an in situ levansucrase assay. Secretion of levansucrase may play an important role for multiplication of E. amylovora in sucrose containing plant tissue.

Solution properties of levan polysaccharide from Pseudomonas syringae pv. phaseolicola, and its possible primary role as a blocker of recognition during pathogenesis

Bacterial levan, a highly branched, high molecular weight polymer of fructose, was purified from culture supernatants of Pseudomonas syringaepv.phaseolicola grown in a liquid high-sucrose medium, and the predominance of β-(2 → 6) linkages was confirmed by 13C NMR. The solution properties of this material resembled those of disordered linear polysaccharides in the response to low-amplitude oscillatory shear (frequency dependence of G′ and G″); the absence of any detectable conformational change with temperature (as monitored by optical rotation); close superposition of steady-shear viscosity (η) and complex dynamic viscosity (/gh∗) at equivalent values of shear-rate (γs−1) and frequency (ωrad s−1); a similar form of shear-thinning (giving linear plots of η versusηγ0·76); and the onset of semi-dilute behaviour at a closely comparable degree of space-occupancy (c[η] ≈ 3·6). The intrinsic viscosity, however, was unusually low ([η] ≈ 0·17 dl g−1) and the concentration dependence of ‘zero-shear’ viscosity in the semi-dilute regime unusually high (η0 ∼ c9·3), as anticipated from the densely packed, branched molecular structure. Solutions of levan and pectin, matched to approximately the same initial viscosity, showed a substantial reduction in viscosity when mixed. Similar behaviour was observed for mixed solutions of levan with locust bean gum (LBG), chosen for its structural similarity to cellulose and hemicelluloses of the plant cell wall. Viscosity reduction was eliminated at low concentrations (indicating that it does not arise from heterologous association), but became very pronounced at high concentrations, and was then accompanied by resolution into levan-rich and LBG-rich solution phases. This evidence of strong thermodynamic incompatibility and exclusion behaviour with (1 → 4)-linked plant polysaccharides suggests that the primary role of levan during pathogenesis may be as a barrier to intimate morphological contact (recognition) between plant cell walls and those of the parasite, thus inhibiting initiation of a hypersensitive response by the host.

Characterization and PCR-based Typing of Xanthomonas campestris pv. vesicatoria from Peppers and Tomatoes in Serbia

During the last two decades bacterial strains associated with necrotic leaf spots of pepper and tomato fruit spots were collected in Serbia. Twenty-eight strains isolated from pepper and six from tomato were characterized. A study of their physiological and pathological characteristics, and fatty acid composition analysis revealed that all of the strains belong to Xanthomonas campestris pv. vesicatoria. Being non-amylolytic and non-pectolytic, pathogenic on pepper but not on tomato, containing lower amounts of fatty acid 15 : 0 ante–iso, the pepper strains were designated as members of the A group of X. campestris pv. vesicatoria. However, the tomato strains hydrolyzed starch and pectate, caused compatible reactions on tomato but not on pepper, had higher percent of 15 : 0 ante–iso fatty acid, and were classified into B phenotypic group and identified as X. vesicatoria. PCR primers were developed which amplified conserved DNA regions related to the hrp genes of different strains of X. campestris pv. vesicatoria associated with pepper and tomato. Restriction analysis of the PCR product resulted in different patterns and enabled grouping of the strains into four groups. When xanthomonads isolated from pepper and tomato in Serbia were analyzed, they clustered into two groups corresponding to the grouping based on their physiological and pathological characteristics. According to the reaction of pepper and tomato differential varieties, the strains from pepper belong to races P7 and P8 and tomato strains belong to the race T2. All strains were sensitive to copper and streptomycin. Advantages and disadvantages of various bacterial spot management practices are discussed.

The role of extracellular polysaccharides as virulence factors for phytopathogenic pseudomonads and xanthomonads

Bacterial exopolysaccharides (EPS) were investigated for their role as virulence factors of leaf spot diseases caused by pseudomonads and xanthomonads. The capacity of these bacteria to induce persistent water-soaking in leaves plays a crucial role during pathogenesis that seems to be accomplished by a synergistic interaction between bacterial EPS and plant polymers. Under conditions of low EPS production (e.g. in continuously darkened plants) the bacteria were not able to cause typical water-soaked disease symptoms. The main EPS components were alginate and levan (Pseudomonas), xanthan (Xanthomonas), as well as lipopolysaccharides (LPS) and a small amount of proteins. It is suggested that alginate which is very similar to plant pectate is required for establishing bacterial infections in later disease stages. This concept was confirmed by evaluating transposon mutants with EPS deficiencies. LPS may be involved in specific interactions with plant polymers leading to agglutination and precipitation (incompatibility) or gel-formation (compatibility). Bacteria which are embedded in a gel-like matrix inplanta are not easily recognized by the plant and are protected against bacteriostatic compounds and desiccation.

The O-chain polysaccharide of the lipopolysaccharide of Xanthomonascampestris pv. begoniae GSPB 525 is a partially l-xylosylated l-rhamnan

The O-chain polysaccharide (OPS) of the lipopolysaccharide of Xanthomonas campestris pv. begoniae GSPB 525 was found to contain L-rhamnose and L-xylose in the ratio 1:0.6. The OPS lacked strict regularity because of nonstoichiometric xylosylation of the main rhamnan chain. Based on methylation analysis, Smith degradation, and 1H and 13C NMR spectroscopy, including COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, the following structure of the OPS was established: [formula: see text].

Development of a new semiselective medium for isolating Xanthomonas campestris pv. manihotis from plant material and soil.

ABSTRACT An effective control for bacterial blight of cassava (Manihot esculenta), caused by Xanthomonas campestris pv. manihotis, requires the use of non-contaminated cuttings and seeds. Using classical agar plating techniques for screening planting material for contamination has not been very successful because of the lack of a reliable semiselective agar medium. The pathogen grows slowly on general plating media and is easily overgrown by saprophytic bacteria during isolation from diseased plants. In an effort to develop a semiselective medium, the utilization of several carbon and nitrogen sources was studied. Results of these tests provided information used to design a basal medium allowing good growth of the target organism while suppressing growth of several common saprophytes. Additional selectivity was achieved by incorporating three antibiotics into the basal medium. The new semiselective agar medium, designated cefazolin trehalose agar (CTA) medium, contained (per liter) 3.0 g of K(2)HPO(4), 1.0 g of NaH(2)PO(4), 0.3 g of MgSO(4).7H(2)O, 1.0 g of NH(4)Cl, 9.0 g of D(+)-trehalose, 1.0 D(+)-glucose, 1.0 g of yeast extract, 0.025 g of cefazolin, 0.0012 g of lincomycin, 0.0025 g of phosphomycin, 0.25 g of cycloheximide, and 14.0 g of agar. In comparison to a starch-based semiselective medium (SXM), plating efficiencies using pure cultures of 10 strains of X. campestris pv. manihotis were significantly higher on CTA, with an average of 85 and 50%, respectively. Likewise, isolation and recovery of X. campestris pv. manihotis from infected cassava leaves and contaminated soil were much higher on CTA than on SXM agar. When X. campestris pv. manihotis occurs in high concentrations in diseased tissue, the standard yeast trehalose glucose agar medium supplemented with 250 mug of cycloheximide per ml appears to be satisfactory. The newly developed CTA medium should prove useful for control strategies to identify and remove infected planting material of cassava, as well as for basic ecological studies of the pathogen.

Structure of the O-specific polysaccharides of the lipopolysaccharides of Xanthomonas campestris pv. vignicola GSPB 2795 and GSPB 2796.

The O-specific polysaccharides of Xanthomonas campestris pv. vignicola GSPB 2795 and GSPB 2796 were studied by sugar and methylation analyses, Smith degradation, ID, 2D 1H and 13C NMR spectroscopy. It was found that the polysaccharides are similar branched D-rhamnans lacking strict regularity, and their structures can be described as follows: [carbohydrate equation: see text] where Rha(v) is present in a non-stoichiometric amount, which varies from strain to strain.

Elimination of bean seed-borne bacteria by thermotherapy and meristem culture

Common and halo blights caused by Xanthomonas campestris pv. phaseoli, and Pseudomonas syringae pv. phaseolicola, respectively, are major world-wide seed-borne diseases of bush bean (Phaseolus vulgaris L.) [1], Seeds contaminated either internally or externally constitute the primary source of the inoculum. In annual plants the main mode of bacterial disease transmission is via the seeds.

Production of the chlorosis inducing toxin in liquid cultures ofPseudomonas phaseolicola (Burkh.) Dowson

Amino acids affected amount and time course of the toxin production on complex as well as on chemically defined media. With glutamic acid the toxin content was high in the early growth stage but decreased later on, while the reverse was true with cysteine as single amino acid.Toxin production was highest at temperatures below 20°C. Generally, the toxin production started during the rapid growth phase and reached its maximum shortly after growth ceased. Chloramphenicolinhibited bacteria did not produce much toxin. Bacterial cells contained relatively low amounts of the toxin. Conditions for toxin production in large quantities are described. The feasibility to produce tritiummarked toxin was studied.Comparison of different strains showed that avirulent or weakly virulent ‘halo-less’ strains do not produce detectable amounts of toxin. However, there was no correlation of the toxin production to the grouping of the bacterial strains in race 1 or the more virulent race 2.

Structures of the O-polysaccharide chains of the lipopolysaccharides of Xanthomonas campestris pv. phaseoli var. fuscans GSPB 271 and X. campestris pv. malvacearum GSPB 1386 and GSPB 2388

O-polysaccharides of phytopathogenic bacteria Xanthomonas campestris were isolated by mild acid degradation of the lipopolysaccharides and studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. The following structures of the repeating units of the polysaccharides of X. campestris pv. phaseoli var. fuscans GSPB 271 (1). and X. campestris pv. malvacearum GSPB 1386 and GSPB 2388 (2). were established:The O-polysaccharides of X. campestris are structurally similar to those of some Pseudomonas syringae strains.