Scanning Electron Microscopy of Invasion of Apple Leaves and Blossoms by Pseudomonas syringae pv. syringae

Scanning electron microscopy indicated that Pseudomonas syringae pv. syringae L795 entered leaves through stomata and multiplied in the substomatal chambers. Strain L195 applied to blossoms colonized stigmas and also occurred in intercellular spaces of styles. Nonpathogenic strain L796 failed to colonize blossoms. This study suggests that inoculum of pathogenic P. syringae pv. syringae builds up on apple leaves and blossoms.     

Systemic Invasion of Immature Sweet Cherry Fruit by Pseudomonas syringae pv. morsprunorum Through Blossoms1

Cherry blossoms inoculated with a rifampicin-resistant strain of Pseudomonas syringae pv. morsprunorum died or gave rise to fruits containing necrotic spots at or near the blossom ends. Scanning electron microscopy of developing fruits indicated that the pathogen had invaded the entire pericarp, including the endocarp. Bacteria also spread to the fruit stalk and, to a lesser extent, to the spurs. Mesocarp cells below the lesion collapsed. Infected fruit, stalks, and spurs contained, respectively, ca. 10⁹, 10⁷, and 10² colony forming units of P. syringae pv. morsprunorum as determined by a dilution plate method on an agar medium supplemented with 50 μg/ml rifampicin. This is the first report of systemic spread of P. syringae from blossoms to developing fruit of a deciduous crop.     

Scanning Electron Microscopy of Olive and Oleander Leaves Colonized by Pseudomonas syringae subsp. Savastanoi

Leaves of olive and oleander were sprayed with suspensions of their homologous strains (PVBa230 and ITM519, respectively) of Pseudomonas syringae subsp. savastanoi and examined by SEM. It was found that both strains multiplied on the lower surface of the leaves of both species. Preferred sites for survival and multiplication were the shields of peltate hairs on olive and the stomatal pits on oleander. The findings suggest that, at least in olive leaves, some bacteria entered the leaf tissue through the stornata but that this was not important since cells which entered the host did not cause any disease symptoms. Cells of both strains were agglomerated and cells of ITM519 were further attached to the surface of the leaf hairs on oleander by fibrillar material. Inoculated leaves did not show any disease symptoms except at leaf abscission scars on olive plants where leaves had been excised prior to inoculation. It is suggested that on both olive and oleander preexisting wounds are necessary for symptoms to develop.     

Transmission Electron Microscopy of Susceptible and Resistant Tomato Leaves Following Infection with Pseudomonas syringae pv. tomato

Ultrastructural changes in tomato leaves of susceptible cv. Peto 95 and resistant cv. Ontario 7710 infected with Pseudomonas syringae pv. tomato were followed by transmission electron microscopy. Up to 48 hours from the inoculation host cells of both cultivars looked quite normal and no bacteria were visible in the intercellular spaces; bacterial cells were found only in the substomatal chambers. Afterwards, the leaf cells of cv. Peto 95 began to degenerate and bacteria invaded the intercellular spaces which seemed enlarged. After 15 days the disorganization was complete: tomato cells were plasmolyzed and the intercellular spaces were filled with bacteria. In the leaves of resistant cv. Ontario 7710 no bacteria were observed later than 48 hours and no visible modifications occurred up to 15 days after the inoculation.     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Structure of the sidechain of lipopolysaccharide from Pseudomonas syringae pv. morsprunorum C28

The sidechain of the lipopolysaccharide from the phytopathogen Pseudomonas syringae pv. morsprunorum C28 was shown to be composed of D-rhamnose. Using 1H and 13C-NMR spectroscopy, methylation analysis, Smith degradation and optical rotation data, the repeat unit was found to have the structure: ----3)-D-Rhap-(alpha 1----3)-D-Rhap-(alpha 1----2)-D-Rhap-(alpha 1---- and a degree of polymerization of approximately 70. Attention is drawn to the possible prevalence of D-6-deoxyhexoses in the lipopolysaccharides of plant pathogenic bacteria.     

Scanning Electron Microscopy of Conidium Formation of Stemphylium vesicarium on Onion Leaves

Onion leaves were inoculated with conidia of Stemphylium vesicarium and the development and morphology of conidiophores and conidia on the leaf surface were examined using scanning electron microscopy. Solitary, but usually fasciculate conidiophores emerged through the epidermis. Hyphae growing on or above the leaf surface also differentiated into conidiophores. Conidiophores were straight or flexuous, simple, smooth or verrucose and cylindrical but enlarged apically at the site of conidiumproduction. Smooth, round, bud-like conidial initials were produced singly at the apex of the verrucose conidiophores. As conidia matured, they became oblong to ovoid and densely verrucose. Once the mature conidium seceded, a small pore was visible at the, apex of the conidiogenous cell. Conidiophores proliferated percurrently at the distal region, forming secondary conidiophores and conidia.     

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

Surface Ultrastructural Studies on Ingress and Establishment of Pseudomonas syringae pv. mori on Mulberry Leaves

P. syringae pv. mori multiplied on leaf surface and colonized particularly on the cystoliths and in the grooves of veins. The masses of bacteria were associated with necrotic spots, which appeared 9 days after inoculation. The studies also revealed that the bacterium invaded leaf tissues through cystoliths. However, it did not enter through stomata and trichomes which had commonly been observed in most of the plant pathogenic bacteria.     

Characterization of alginate lyase from Pseudomonas syringae pv. syringae

The gene encoding alginate lyase (algL) in Pseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42 degrees C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.     

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from 1D and 2D 1H- and 13C-NMR spectra combined with extensive FAB-MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodepsinonapeptide. Its peptide moiety corresponds to Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acetylated by 3-hydroxytetradecanoic acid.     

Chemotaxis by Pseudomonas syringae pv. tomato

Optimal laboratory conditions for studying chemotaxis by Pseudomonas syringae pv. tomato were determined by using the Adler capillary tube assay. Although they are not an absolute requirement for chemotaxis, the presence of 0.1 mM EDTA and 1 mM MgCl₂ in the chemotaxis buffer (10 mM potassium phosphate [pH 7.2]) significantly enhanced the response to attractant. The addition of mannitol as an energy source had little effect. The optimal temperature for chemotaxis was 23°C, which is 5°C below the optimal growth temperature for this pathogen. The best response occurred when the bacteria were exposed to attractant for 60 min at a concentration of approximately 5 × 10⁶ CFU/ml. P. syringae pv. tomato was strongly attracted to citric and malic acids, which are the predominant organic acids in tomato fruit. With the exception of asparagine, the major amino acids of tomatoes were weak to moderate attractants. Glucose and fructose, which account for approximately 47% of tomato dry matter, also elicited poor responses. In assays with tomato intercellular fluid and leaf surface water, the bacterial speck pathogen could not chemotactically distinguish between a resistant and a susceptible cultivar of tomato.     

Characterization of Pyoverdinpss, the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin_pss, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin_pss of approximately 50 μg/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of ≥ 10 μM. Pyoverdin_pss was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin_pss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin_pss was structurally unique since amino acid analysis of reductive hydrolysates yielded β-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin_pss exhibited a relatively high affinity constant for Fe(III), with values of 10²⁵ at pH 7.0 and 10³² at pH 10.0. Iron uptake assays with [⁵⁵Fe]pyoverdin_pss demonstrated rapid active uptake of ⁵⁵Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin_pss. The chemical and biological properties of pyoverdin_pss are discussed in relation to virulence and iron uptake during plant pathogenesis.     

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.     

Analysis of Sweet cherry (Prunus avium L.) Leaves for Plant Signal Molecules That Activate the syrB Gene Required for Synthesis of the Phytotoxin,Syringomycin, by Pseudomonas syringae pv syringae

An important aspect of the interaction of Pseudomonas syringae pv syringae with plant hosts is the perception of plant signal molecules that regulate expression of genes, such as syrB, required for synthesis of the phytotoxin, syringomycin. In this study, the leaves of sweet cherry (Prunus avium L.) were analyzed to determine the nature of the syrB-inducing activity associated with tissues of a susceptible host. Crude leaf extracts yielded high amounts of total signal activity of more than 12,000 units g-1 (fresh weight) based on activation of a syrB-lacZ fusion in strain B3AR132. The signal activity was fractionated by C18 reversed-phase high-performance liquid chromatography and found to be composed of phenolic glycosides, which were resolved in three regions of the high-performance liquid chromatography profile, and sugars, which eluted with the void volume. Two flavonol glycosides, quercetin 3-rutinosyl-4[prime]-glucoside and kaempferol 3-rutinosyl-4[prime]-glucoside, and a flavanone glucoside, dihydrowogonin 7-glucoside, were identified. The flavonoid glycosides displayed similar specific signal activities and were comparable in signal activity to arbutin, a phenyl [beta]-glucoside, giving rise to between 120 and 160 units of [beta]-galactosidase activity at 10 [mu]M. Although D-fructose exhibits intrinsic low level syrB-inducing signal activity, D-fructose enhanced by about 10-fold the signal activities of the flavonoid glycosides at low concentrations (e.g. 10 [mu]M). This demonstrates that flavonoid glycosides, which represent a new class of phenolic plant signals sensed by P. s. syringae, are in sufficient quantities in the leaves of P. avium to activate phytotoxin synthesis.     

Some characteristics of Pseudomonas syringae pv. maculicola dissociants

Pseudomonas syringae pv. maculicola dissociants producing colonies of different morphotype were found to possess similar biochemical and serological properties but different virulence to the host plant. The heterogeneous extracellular and intracellular lipopolysaccharide-protein complexes of the dissociants differed in their chemical composition and biological activity towards test plants.