High-performance liquid chromatography analyses of pyoverdin siderophores differentiate among phytopathogenic fluorescent Pseudomonas Species

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two beta-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.     

Pseudomonas syringae pv. japonica (Mukoo 1955) Dye et al. 1980 is a junior synonym of Ps. syringae pv. syringae van Hall 1902

An investigation of the biochemical, nutritional and pathogenic reactions of strains of Pseudomonas syringae pv. japonica and Ps. syringae pv. syringae showed them to be indistinguishable. Pseudomonas syringae pv. japonica is a junior synonym of Ps. syringae pv. syringae.     

Genetic characterization of Pseudomonas 'NZI7'--a novel pathogen that results in a brown blotch disease of Agaricus bisporus

AIMS: To characterize a novel pseudomonad isolate capable of causing brown blotch disease of Agaricus bisporus. METHODS AND RESULTS: Using the white-line-in-agar (WLA) assay, fluorescent pseudomonads isolated from a New Zealand mushroom farm were screened for the lipodepsipeptide tolaasin, a characteristic marker of Pseudomonas tolaasii. One isolate, NZI7, produced a positive WLA assay and caused brown lesions of A. bisporus comparable with those produced by Ps. tolaasii. However, genetic analysis suggested that Ps. tolaasii and NZI7 were genetically dissimilar, and that NZI7 is closely related to Pseudomonas syringae. Nucleotide sequence analyses of a gene involved in tolaasin production indicated that similar genes are present in both NZI7 and Ps. tolaasii. CONCLUSION: NZI7 represents a novel Pseudomonas species capable of causing brown blotch disease of A. bisporus. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic identification of Ps. tolaasii based on A. bisporus browning and positive WLA may have limited specificity.     

Isolation and Characterization of the algD gene of Pseudomonas syringae pv. phaseolicola and its distribution among other Pseudomonads and related Organisms

The gene coding for GDP-mannose dehydrogenase ( algD ) was isolated from a Pseudomonas syringae pv. phaseolicola genomic library using a polymerase chain reaction-generated heterologous DNA-probe from Pseudomonas aeruginosa . A total of 2123 base pairs were sequenced (accession number AF001555) and analysed for homologies to the alginate gene cluster of P. aeruginosa . Downstream from algD an alg8 homologue was found suggesting a similar arrangement of the alginate gene cluster in P. syringae pv. phaseolicola to that in P. aeruginosa . Also, the deduced amino acid sequence of algD shows high similarity to that of P. aeruginosa (0.9) and Azotobacter vinelandii (0.88). Southern hybridization experiments revealed that algD is widely distributed among members of the Pseudomonas rRNA homology group I. Among others, sequences homologous to algD were detected in the P. syringae pathovars lachrymans , mori , morsprunorum, pisi , savastanoi, tabaci and tomato as well as in Pseudomonas amygdali . For most of the algD positive organisms synthesis of alginate has been reported by other studies. However, algD homologues were also detected for the species Pseudomonas corrugata , Pseudomonas marginalis and Pseudomonas avenae ( Acidovorax avenae ), for which alginate biosynthesis has not yet been reported.     

Serological and molecular size characterization of flagellins of Pseudomonas syringae pathovars and related bacteria

Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P. chlororaphis, P. cichorii, P. corrugata, P. fluorescens, P. fuscovaginae, P. stutzeri, P. viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting. Eighty-six strains of the P. syringae group pathovars, P. cichorii and P. viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa. Similarities between the P. syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out. The distribution of H1 and H2 antigens in the nine recently described genomospecies of P. syringae-P. viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads. The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.     

Identification of Pseudomonas tolaasi: the White Line in Agar and Mushroom Tissue Block Rapid Pitting Tests

A sharply defined white line of precipitate forms in Pseudomonas Agar F (Difco) between the opaque white colonies of Pseudomonas tolaasi and translucent colonies of certain unidentified pseudomonads. This visible interaction has been utilized in a specific and reliable method for the identification of Ps. tolaasi. The white line test was positive when 113 isolates of Ps. tolaasi from five different countries were examined, whereas 154 isolates of pseudomonads other than Ps. tolaasi , including Ps. corrugata, Ps. delphinii, Ps. fluorescens, Ps. lachrymans, Ps. marginalis, Ps. pastinaceae, Ps. phaseolicola, Ps. aeruginosa, Ps. putida, Ps. syringae, Ps. mors-prunorum, Ps. cichorii, Ps. antirrhini, Ps. viridiflava, Ps. caryophylli, Ps. cepacia, Ps. mendocina, Ps. stutzeri, Ps. acidivorus and Ps. lemoignei did not give the white line reaction with a reacting translucent colony pseudomonad. Browning of mushrooms in host tests does not help in the identification of Ps. tolaasi , but a conspicuous pitting produced in less than 10 min at the cut surface of mushroom tissue is as specific as the white line test in detecting Ps. tolaasi in suspension in distilled water.     

Detection of tabtoxin-producing strains of Pseudomonas syringae by PCR

AIMS: The present study describes a system based on PCR to distinguish tabtoxin-producing strains of Pseudomonas syringae from other Ps. syringae plant pathogens that produce chlorosis-inducing phytotoxins. METHODS AND RESULTS: Thirty-two strains of Ps. syringae and related species were examined. Two sets of PCR primers were developed to amplify genes (tblA and tabA) required for tabtoxin production. Only a PCR product of 829 bp or 1020 bp was produced in PCR reactions with the tblA or tabA primer sets, respectively, and cells from tabtoxin-producing pathovars of Pseudomonas syringae. All known non-tabtoxin producing bacterial species failed to produce an amplification product with either primer set. CONCLUSIONS: PCR of genes required for tabtoxin production is a simple, rapid and reliable method for identifying tabtoxin-producing strains of Ps. syringae. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol can effectively distinguish tabtoxin-producing strains of Ps. syringae from other Ps. syringae pathovars and Ps. syringae pv. tabaci strains from other tabtoxin-producing Ps. syringae pathovars.     

Influence of subculturing some phytopathogenic bacteria on their carbohydrate utilizing profile

The capability of carbohydrate utilization shown by some freshly-isolated phytopathogenic bacteria (i.e. Pseudomonas syringae pv. syringae, Ps.s. pv. actinidiae, Ps.s. pv. lachrymans Ps. cichorii, Xanthomonas campestris pv. campestris ) was assessed by means of the API system and compared with that shown by the same isolates when they were subsequently subcultured for I week and for 1 month on nutrient agar and on the medium B of King et al. All isolates showed a differential capability of utilization during the subcultures of the laboratory culture media. In some cases, the utilization of some carbohydrates was not shown during the in vitro growth. On the other hand, the life on bacterial culture media induced the capability to utilize other carbohydrates. The need for standardization of recent techniques for the assessment of organic compound utilization is stressed.     

Biofilm formation and cellulose expression among diverse environmental Pseudomonas isolates

The ability to form biofilms is seen as an increasingly important colonization strategy among both pathogenic and environmental bacteria. A survey of 185 plant-associated, phytopathogenic, soil and river Pseudomonas isolates resulted in 76% producing biofilms at the air-liquid (A-L) interface after selection in static microcosms. Considerable variation in biofilm phenotype was observed, including waxy aggregations, viscous and floccular masses, and physically cohesive biofilms with continuously varying strengths over 1500-fold. Calcofluor epifluorescent microscopy identified cellulose as the matrix component in biofilms produced by Pseudomonas asplenii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas putida, Pseudomonas savastanoi and Pseudomonas syringae isolates. Cellulose expression and biofilm formation could be induced by the constitutively active WspR19 mutant of the cyclic-di-GMP-associated, GGDEF domain-containing response regulator involved in the P. fluorescens SBW25 wrinkly spreader phenotype and cellular aggregation in Pseudomonas aeruginosa PA01. WspR19 could also induce P. putida KT2440, which otherwise did not produce a biofilm or express cellulose, as well as Escherichia coli K12 and Salmonella typhimurium LT2, both of which express cellulose yet lack WspR homologues. Statistical analysis of biofilm parameters suggest that biofilm development is a more complex process than that simply described by the production of attachment and matrix components and bacterial growth. This complexity was also seen in multivariate analysis as a species-ecological habitat effect, underscoring the fact that in vitro biofilms are abstractions of those surface and volume colonization processes used by bacteria in their natural environments.     

PCR assays for specific and sensitive detection of Pseudomonas tolaasii,the cause of brown blotch disease of mushrooms

AIMS: The present study describes PCR assays to detect specifically Pseudomonas tolaasii from various samples. METHODS AND RESULTS: Two sets of PCR primers were developed to amplify genes required for tolaasin production. Only a PCR product of 449 bp or 249 bp was produced in PCR reactions with the Pt-1A/Pt-1D1 or Pt-PM/Pt-QM primer sets, respectively, and DNA and cells of Ps. tolaasii. Nested and immunocapture-nested PCR could detect to 3 cells of Ps. tolaasii and amplify the Ps. tolaasii-specific DNA from a sample containing 10 000 times more other bacterial cells than Ps. tolaasii, respectively. CONCLUSIONS: The PCR assays are simple, rapid and reliable methods for detection and identification of Ps. tolaasii. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocols can effectively distinguish Ps. tolaasii from other bacteria and detect Ps. tolaasii from various samples for studying ecology of the bacterium and preventing the use of contaminated water or spawn or medium in mushroom cultivation.     

Protein and enzymatic criteria for the characterization of phytopathogenic Pseudomonas fluorescens

Polyacrylamide gel electrophoresis of proteins was carried out to characterize eight bacterial strains belonging to the genus Pseudomonas. The sampling included three species (P. cichorii, P. viridiflava and P. syringae), with three pathovars for this last species (pv. pisi, pv. syringae, pv. tomato). Several molecular markers were evaluated: native proteins, denatured proteins, esterases, superoxide dismutases (SOD) and polyphenoloxidases (PPO). Each species or pathovar of Pseudomonas was clearly differentiated by esterase patterns. SOD, PPO and native protein patterns allowed strains of P. cichorii, P. viridiflava and P.s. pv. tomato also to be distinguished. Strains of P.s. pv. pisi and P.s. pv. syringae were identical for these criteria. Denatured protein patterns of these two pathovars and P. viridiflava were similar.     

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.     

Evaluation of determinative tests for pathovars of Pseudomonas syringae van Hall 1902

The utility of 36 presumptive determinative tests for 32 pathovars of Pseudomonas syringae was investigated. A total of 395 strains was examined. Most strains of 12 of these pathovars ( Ps. syringae pv. cannabina, Ps. syr. delphinii, Ps. syr. glycinea, Ps. syr. helianthi, Ps. syr. lachrymans, Ps. syr. mori, Ps. syr. morsprunorum, Ps. syr. phaseolicola, Ps. syr. 'porri', Ps. syr. papulans, Ps. syr. savastanoi and Ps. syr. tabaci ) formed clusters when test data were compared by centroid analysis. Pseudomonas syr. syringae, Ps. syr. aptata, Ps. syr. atrofaciens, Ps. syr. dysoxyli and Ps. syr. japonica formed a single cluster, indicating their possible synonymy. Strains of Ps. syr. antirrhini and Ps. syr. tomato were indistinguishable, as were those of Ps. syr. garcae and Ps. syr. oryzae. Strains of Ps. syr. berberidis, Ps. syr. coronafaciens, Ps. syr. eriobotryae, Ps. syr. maculicola, Ps. syr. passiflorae, Ps. syr. pisi and Ps. syr. striafaciens and Ps. syr. tagetis did not form distinguishable clusters.
The tests which reliably differentiated pathovars are recorded in a determinative scheme.     

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.     

Bacteriocin production by Pseudomonas syringae pv. ciccaronei NCPPB2355. Isolation and partial characterization of the antimicrobial compound

Pseudomonas syringae pv. ciccaronei strain NCPPB2355 was found to produce a bacteriocin inhibitory against strains of Ps. syringae subsp. savastanoi , the causal agent of olive knot disease. Treatments with mitomycin C did not substantially increase the bacteriocin titre in culture. The purification of the bacteriocin obtained by ammonium sulphate precipitation of culture supernatant fluid, membrane ultrafiltration, gel filtration and preparative PAGE, led to the isolation of a high molecular weight proteinaceous substance. The bacteriocin analysed by SDS-PAGE revealed three protein bands with molecular weights of 76, 63 and 45 kDa, respectively. The bacteriocin was sensitive to heat and proteolytic enzymes, was resistant to non-polar organic solvents and was active between pH 5·0–7·0. Plasmid-DNA analysis of Ps. syringae ciccaronei revealed the presence of 18 plasmids; bacteriocin-negative variants could not be obtained by cure experiments.     

Siderophore typing,a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.     

Isozyme analysis for the identification of Pseudomonassyringae pathovar pisi strains

Distinction between Pseudomonas syringae pathovar (pv.) pisi (Ps. syr. pisi) , responsible for bacterial blight of pea ( Pisum sativum ), and pv. syringae (Ps. syr. syringae) , still requires strain inoculation onto peas. Patterns of enzymes including esterase (EST) and superoxide dismutase (SOD) were examined for diagnostic purposes. Profiles of 59 Ps. syr . pisi strains and 53 Ps. syr . syringae strains were compared. Pseudomonas syringae pisi was characterized by one unique zymotype for SOD and two slightly different zymotypes for EST. Pseudomonas syringae syringae zymotypes were very heterogeneous with 10 different zymotypes for SOD and 32 for EST. Twenty-four percent of the Ps. syr . syringae strains shared SOD zymotype 1 of Ps. syr . pisi , thus preventing the use of this enzymatic system for identification. In contrast, the two EST zymotypes of Ps. syr. pisi strains were specific to the pathovar and could be used for its identification. The two Ps. syr. pisi EST patterns were correlated to race structure of the pathovar, zymotype 1 corresponding to races 2, 3, 4 and 6, and zymotype 2 to races 1, 5 and 7. Esterase isozyme profiling was proposed as a new identification procedure for bacterial pea blight agent.     

The specificity of an immune serum to the exocellular lipopolysaccharide of Pseudomonas wieringae

The serum obtained to exocellular lipopolysaccharide (ELPS) of Pseudomonas wieringae selectively agglutinated strains of pathovar of P. syringae and did not agglutinated strains of P. cichorii, P. solanacearum, P. gladioli pv. allicola, P. fluoroviolaceus, strains of nonphytopathogenic pseudomonads as well as bacteria of the genera Erwinia, Bacillus, Xanthomonas, Klebsiella. Consequently, the antigen determinant common with antigen of the species Pseudomonas syringae is present in the composition of ELPS.     

Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple

Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, non-fluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10(-1)-10(-2) per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10(-2) to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3.9 kb EcoRI and 3.0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3.9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.     

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.