Induction of Resistance or Susceptibility in Pea to Races of Pseudomonas syringae pv. pisi

The phenomena of induced resistance and induced susceptibility were investigated in the pea-Pseudomonas syringae pv. pisi system, using two pea cultivgars, Early Onward and Hurst Green Shaft, and races 1 and 2 of the pathogen. Preliminary treatment with heat-killed bacteria induced resistance in peas to infection by P. s. pv. pisi; the resistance induced was dependent on the time interval between the preliminary and challenge inoculations. The mechanism of induced resistance appears to vary between the cultivars. Similarly, both races of the pathogen appear to have different resistance-inducing efficiencies. Resistance in cvs Early Onward and Hurst Green Shaft to the compatible bacterium could not be induced by preliminary inoculation with live cells of an incompatible race. Heat-killed cells of the races failed to induce the hypersensitive reaction in cultivars that normally show this response when challenged with live avirulent bacteria. Preliminary inoculation with live race 1 cells failed to induce susceptibility in cv. Early Onward to live race 2 cells, irrespective of the challenge inoculation interval. On cv. Hurst Green Shaft, however, preliminary inoculation with live race 2 cells induced limited susceptibility to live race 1 cells. Preliminary treatment with sterile distilled water followed by challenge of the same leaves 24 h later with a compatible race induced a moderate resistance response in both cultivars.     

Induction of Resistance or Susceptibility in Pea to Races of Pseudomonas syringae pv. pisi

The phenomena of induced resistance and induced susceptibility were investigated in the pea-Pseudomonas syringae pv. pisi system, using two pea cultivars, Early Onward and Hurst Green Shaft, and races 1 and 2 of the pathogen. Preliminary treatment with heat-killed bacteria induced resistance in peas to infection by P. s. pv. pisi; the resistance induced was dependent on the time interval between the preliminary and challenge inoculations. The mechanism of induced resistance appears to vary between the cultivars. Similarly, both races of the pathogen appear to have different resistance-inducing efficiencies. Resistance in cvs Early Onward and Hurst Green Shaft to the compatible bacterium could not be induced by preliminary inoculation with live cells of an incompatible race. Heat-killed cells of the races failed to induce the hypersensitive reaction in cultivars that normally show this response when challenged with live avirulent bacteria. Preliminary inoculation with live race 1 cells failed to induce susceptibility in cv. Early Onward to live race 2 cells, irrespective of the challenge inoculation interval. On cv. Hurst Green Shaft, however, preliminary inoculation with live race 2 cells induced limited susceptibility to live race 1 cells. Preliminary treatment with sterile distilled water followed by challenge of the same leaves 24 h later with a compatible race induced a moderate resistance response in both cultivars.     

Interaction between Pseudomonas syringae pv. Pisi and Isolated Pea Mesophyll Protoplasts

The interaction between five races of Pseudomonas syringae pv. pisi (PSP) and isolated mesophyll protoplasts obtained from five pea cultivars was studied. There was no trend in the attachment of bacterial cells to surfaces of compatible and incompatible host protoplasts. The viability of protoplasts from compatible and incompatible host-pathogen interactions did not differ significantly; however, changes in the viability of cultivar Kelvedon Wonder protoplasts, compatible with all five races, was relatively more stable following inoculation with bacteria than those of cultivar Fortune, incompatible with all of the five races. Protoplast cell wall regeneration did not take place until 24–48 h after isolation. It is concluded that the use of pea protoplasts as a model system for studying the pea-PSP interaction appears to have considerable potential for the future, but more basic research is required.     

Isolation of Race-and Cultivar-Specific Elicitors from Intercellular Fluids of Compatible Interactions of Pseudomonas syringae pv. pisi and Pea

Intercellular fluids obtained by an in vacuo infiltration technique from compatible race-cultivar interactions of five races of Pseudomonas syringae pv. pisi and pea induced extensive light brown necrotic (hypersensitive type of) lesions in resistant but not susceptible cultivars. In susceptible cultivars the intercellular fluids induced extetensive water soaking symptoms. The intercellular fluids elicited intermediate reactions in pea cultivars of moderate resistance. The intensity of the light brown necrotic reactions in pea appears to be positively correlated with the degree of resistance.     

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.     

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.     

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.     

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.     

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.     

Biological Control of Pseudomonas syringae pv. glycinea by Epiphytic Bacteria under Field Conditions

The efficacy of a bacterial strain as a biocontrol agent in the field may be related to the ecological similarity between the biocontrol agent and the target pathogen. Therefore, a number of different Pseudomonas syringae strains were evaluated for their antagonistic activities in vitro (agar-diffusion assay) and in planta (greenhouse assay) against the target pathogen, Pseudomonas syringae pv. glycinea. Six strains of five different pathovars were found to be antagonistic in vitro as well as in planta. The epiphytic fitness of the antagonistic Pseudomonas syringae strain 22d/93 and its two antibiotic-resistant mutants were examined on soybean plants in the fields. After adaptation the parental strain and its mutants had the ability to establish and maintain large epiphytic populations (about 10⁶ cfu/g FW) over the whole growing season after a single spray inoculation. The epiphytic behaviors of the mutants and the parent were not significantly different. The introduced bacteria did not influence the total bacterial population size. When the antagonist was coinoculated with the pathogen, the development of the pathogen was significantly reduced during the whole growing season. When the antagonistic strain was inoculated 4 weeks in advance of the pathogen, this antagonistic effect could be markedly enhanced. The final population size of the pathogen reached just 10⁴ cfu/g FW and was significantly reduced to 0.12% compared to the pathogen alone. This study demonstrates that biological control of foliar pathogens through colonization of the host plants with near isogenic or ecologically similar antagonistical strains seems to be a realistic goal.     

Effect of pea bacterial blight (Pseudomonas syringae pv. pisi) on the yield of spring sown combining peas (Pisum sativum)

Field trials to examine the effect of pea bacterial blight (Pseudomonas syringae pv. pisi) (Psp) on the yield of combining peas were carried out at five sites (HRI Wellesbourne, ADAS Rosemaund, ADAS Terrington, PGRO, SASA East Craigs) in the UK in 1990, 1991 and 1992. Healthy seed, cv. Solara, and seed naturally infected with Psp Race 2 was sown in large plots (c. 200 m²) in the open or under nets to prevent bird damage by pigeons. Despite relatively low disease severity levels (< 15% leaf area) and separation by at least 12 m of cv. Consort (resistant to Race 2) between plots there was considerable spread of disease into plots sown with healthy seed. Regression analysis demonstrated a significant relationship between yield and disease. Of the disease measurements examined, disease severity on the leaves (stipules) at growth stage 208 was found to be the best predictor of yield. A model containing environmental and bird damage terms, in addition to disease, suggested that a yield loss of 0.98 t/ha would be expected for a disease severity score of 1, equivalent to 5% leaf area affected, at growth stage 208.     

Exopolysaccharides of the phytopathogen Pseudomonas syringae pv. glycinea.

Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS-dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division. This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content. Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments). Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC. In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production.     

Molecular and Physiological Characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola Strains That Produce the Phytotoxin Coronatine

The chlorosis-inducing phytotoxin coronatine is produced by several Pseudomonas syringae pathovars, including glycinea, morsprunorum, atropurpurea, and the closely related tomato and maculicola. To date, all coronatine-producing pv. glycinea, morsprunorum, and atropurpurea strains that have been examined carry the gene cluster that controls toxin production on a large plasmid. In the present study the genomic location of the coronatine gene cluster was determined for coronatine-producing strains of the pv. tomato-maculicola group by subjecting their genomic DNA to pulsed-field electrophoresis and Southern blot analysis with a hybridization probe from the coronatine gene cluster. The cluster was chromosomally borne in 10 of the 22 strains screened. These 10 strains infected both crucifers and tomatoes but could not use sorbitol as a sole source of carbon. The remaining 12 coronatine-producing strains had plasmid-borne toxin gene clusters and used sorbitol as a carbon source. Only one of these strains was pathogenic on both crucifers and tomatoes; the remainder infected just tomatoes. Restriction fragment length polymorphism analysis of the pv. tomato-maculicola coronatine gene clusters was performed with probes from P. syringae pv. tomato DC3000, a tomato and crucifer pathogen. Although the coronatine cluster appeared, in general, to be highly conserved across the pv. tomato-maculicola group, there were significant differences between plasmid-borne and chromosomally borne genes. The extensively studied coronatine cluster of pv. glycinea 4180 closely resembled the plasmid-borne clusters of the pv. tomato-maculicola group.     

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.     

Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae.

One of the chromosomal regions of Pseudomonas syringae pv. syringae encoding pathogenicity factors had been mapped into a 3.9-kilobase-pair fragment in previous studies. Promoter probe analysis indicated the existence of a promoter near one end of the fragment. DNA sequencing of this fragment revealed the existence of a consensus promoter sequence in the region of the promoter activity and two open reading frames (ORFs) downstream. These ORFs, ORF1 and ORF2, encoded putative polypeptides of 40 and 83 kilodaltons, respectively. All ORF1::Tn5 as well as ORF2::Tn5 mutant strains were nonpathogenic on susceptible host bean plants and were unable to elicit hypersensitive reactions on nonhost tobacco plants. The deduced amino acid sequence of the 83-kilodalton polypeptide contained features characteristic of known integral membrane proteins. Fusion of the lacZ gene to ORF2 led to the expression of a hybrid protein inducible in Escherichia coli. The functions of the putative proteins encoded by ORF1 and ORF2 are unknown at present.     

Structure of the Lipopolysaccharide Side-chain of Pseudomonas syringae pv. syringae Strain S29

Using ¹H‐ and ¹³C‐nuclear magnetic resonance spectroscopy, the repeat unit of the lipopolysaccharide side‐chain from Pseudomonas syringae pv. syringae strain S29 was shown to have the following structure: This structure is identical with that of the side‐chain of Pseudomonas syringae pv. mori CFPB 1656. a     

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.