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.     

Variation within Pseudomonas syringae pv. philadelphi, the cause of a leaf spot of Philadelphus spp.

R oberts , S.J. 1985. Variation within Pseudomonas syringae pv. philadelphi , the cause of a leaf spot of Philadelphius spp. Journal of Applied Bacteriology 59 , 283–290
In pathogenicity tests on Philadelphus and other plant species, belonging to ten genera in seven families, isolates of Pseudomonas syringae from leaf spots on Philadelphus spp. in England did not produce symptoms on any plants other than Philadelphus . It is therefore proposed that these isolates should be designated a distinct pathovar of Ps. syringae with the name Pseudomonas syringae pv. philadelphi . Isolates of this new pathovar varied in their reactions to 6 of 57 biochemical tests. In phage typing tests isolates also varied in their sensitivity to five of seven bacteriophage strains. Four of the six biochemical tests (aesculin hydrolysis, utilization of DL-homoserine L-leucine and sorbitol) and all five of the phages (P11, Pls, P2, A15, and A26) were used to separate the isolates into seven groups. These groups had some relation to their geographical origin, species of Philadelphus from which they were originally isolated, and relative virulence on P. coronarius and P. x purpureo-maculatus . They may represent ecotypes of this new pathovar.     

Composition, structure, and biological properties of lipopolysaccharides from different strains of Pseudomonas syringae pv. atrofaciens

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C10:0, C12:0, 2-HO-C12:0, 3-HO-C12:0, C16:0, C16:1, C18:0, and C18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L- (two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substitute. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.     

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.     

A Note: Serological study of four pathovars of Pseudomonas syringae: Ps. syr. aptata, Ps. syr. tabaci, Ps. syr. mors-prunorum and Ps. syr. phaseolicola

The antigenic reactions of 35 strains of four pathovars of Pseudomonas syringae (Ps. syr. aptata, Ps. syr. tabaci, Ps. syr. mors-prunorum and Ps. syr. phaseolicola ) were studied by double diffusion and indirect immunofluorescent staining, and anti-whole-cell and anti-LPS-extract sera. It had already been shown that the precipitating lines in Ouchterlony double-diffusion tests, due to bacterial LPS, were suitable for the distinction of O-serogroups. The investigation of serological cross-reactions between the 35 strains and 20 antisera revealed that three pathovars were serologically homogeneous: Ps. syr. aptata, Ps. syr. tabaci and Ps. syr. phaseolicola. They could fit into three O-serogroups formerly described: namely APTPIS, TAB and PHA. The O-serogroups APTPIS and TAB showed some common antigens. The 10 strains of Ps. syr. mors-prunorum studied were distributed into two O-serogroups (eight strains belonging to the O-serogroup MOP1, one strain to MOP2, and the last strain failed to react with any of the serogroups).     

Structure of the O-polysaccharide of the lipopolysaccharide of Pseudomonas syringae pv. garcae ICMP 8047.

The composition and structure of the O-polysaccharide of the lipopolysaccharide of Pseudomonas syringae pathovar garcae ICMP 8047 were studied using methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The polysaccharide was found to contain L-rhamnose and 3-acetamido-3, 6-dideoxy-D-galactose (D-Fuc3NAc) in the ratio 4:1 and to consist of two types of pentasaccharide repeating units. The major (1) and minor (2) repeating units differ from each other only in the position of substitution of one of the rhamnose residues in the main chain. Similar structural heterogeneity has been reported formerly in O-polysaccharides of some other P. syringae strains having a similar monosaccharide composition. A Fuc3NAc residue is attached to the main rhamnan chain as a side chain by a (alpha1-->4) glycosidic linkage; this has not hitherto been described in P. syringae: [figure].     

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.     

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.     

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.     

Serological heterogeneity of Pseudomonas syringae pv. atrofaciens strains and their ecological niches

The paper deals with a comparative analysis of the serological and ecological properties of Pseudomonas syringae pv. atrofaciens strains from the collections of microbial cultures at the Malkov Institute for Plant Genetic Resources and Zabolotny Institute of Microbiology and Virology. All of the strains from the Bulgarian collection, except for one, fall into five serogroups (II through VI) of the classification system of Pastushenko and Simonovich. The P. syringae pv. atrofaciens strains isolated from Bulgarian and Ukrainian wheats belong mainly to serogroups II and IV, respectively. The strains that were isolated from rye plants belong to serogroup I. The strains isolated from sorghum and Sudan grass belong to serogroups II, IV, and VL. Serogroup III includes the P. syringae pv. atrofaciens strains that were isolated from cereals in the United Kingdom but not in Ukraine.     

Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli

The production of monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (sodium salt) soluble antigens of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli (fuscans strain) is described. MAbs A6-1 and A6-2 produced to Ps. syringae pv. phaseolicola are pathovar specific. Although MAb XP2 produced to X. campestris pv. phaseoli recognized surface antigens of all strains of this pathovar (including fuscans strains) it cross-reacted specifically with X. campestris pv. malvacearum; it did not react with any other known bacteria or unidentified epiphytes from navy bean seed or leaves. The isotype of both MAbs XP2 and A6-1 is IgG3 whereas that of MAb A6-2 is IgG2a. The reactive antigens are thermostable, but their chemical nature has not been determined.     

Prevalence of Pseudomonas aeruginosa O serogroups

The serological typing of 708 P. aeruginosa strains made it possible to determine serogroups in 97.9% of cultures. Serogroups O2 and O6 were the most prevalent (33.8% and 2.5% respectively); serotypes O1, O3 and O11 also occurred rather frequently (about 10%); O4, O7 and O9 were rare (3-8%), serotypes O10 and O12, very rare (less than 1%). The prevalence of P. aeruginosa strains O2 and O6 among the clinical strains was shown over a period of 10 years, serogroup O2 always playing the leading role. In serogroups, the predominance of strains with a definite combination of partial antigen was established; strains with the antigenic structure not described in the International Scheme of the Structure of O-Antigens were detected.     

Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products.

Production of the chlorosis-inducing phytotoxin coronatine in the Pseudomonas syringae pathovars atropurpurea, glycinea, maculicola, morsprunorum, and tomato has been previously reported. DNA hybridization studies previously indicated that the coronatine biosynthetic gene cluster is highly conserved among P. syringae strains which produce the toxin. In the present study, two 17-bp oligonucleotide primers derived from the coronatine biosynthetic gene cluster of P. syringae pv. glycinea PG4180 were investigated for their ability to detect coronatine-producing P. syringae strains by PCR analysis. The primer set amplified diagnostic 0.65-kb PCR products from genomic DNAs of five different coronatine-producing pathovars of P. syringae. The 0.65-kb products were not detected when PCR experiments utilized nucleic acids of nonproducers of coronatine or those of bacteria not previously investigated for coronatine production. When the 0.65-kb PCR products were digested with ClaI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P. syringae. A restriction fragment length polymorphism was detected in the amplified region of P. syringae pv. atropurpurea, since this pathovar lacked a conserved PvuI site which was detected in the PCR products of the other four pathovars. The 0.65-kb PCR products from six strains comprising five different pathovars of P. syringae were cloned and sequenced. The PCR products from two different P. syringae pv. glycinea strains contained identical DNA sequences, and these showed relatedness to the sequence obtained for the pathovar morsprunorum. The PCR products obtained from the pathovars maculicola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Housekeeping gene sequencing and multilocus variable-number tandem-repeat analysis to identify subpopulations within Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. tomato that correlate with host specificity

Pseudomonas syringae pv. maculicola causes bacterial spot on Brassicaceae worldwide, and for the last 10 years severe outbreaks have been reported in the Loire Valley, France. P. syringae pv. maculicola resembles P. syringae pv. tomato in that it is also pathogenic for tomato and causes the same types of symptoms. We used a collection of 106 strains of P. syringae to characterize the relationships between P. syringae pv. maculicola and related pathovars, paying special attention to P. syringae pv. tomato. Phylogenetic analysis of gyrB and rpoD gene sequences showed that P. syringae pv. maculicola, which causes diseases in Brassicaceae, forms six genetic lineages within genomospecies 3 of P. syringae strains as defined by L. Gardan et al. (Int. J. Syst. Bacteriol. 49[Pt 2]:469-478, 1999), whereas P. syringae pv. tomato forms two distinct genetic lineages. A multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) conducted with eight minisatellite loci confirmed the genetic structure obtained with rpoD and gyrB sequence analyses. These results provide promising tools for fine-scale epidemiological studies on diseases caused by P. syringae pv. maculicola and P. syringae pv. tomato. The two pathovars had distinct host ranges; only P. syringae pv. maculicola strains were pathogenic for Brassicaceae. A subpopulation of P. syringae pv. maculicola strains that are pathogenic for Pto-expressing tomato plants were shown to lack avrPto1 and avrPtoB or to contain a disrupted avrPtoB homolog. Taking phylogenetic and pathological features into account, our data suggest that the DC3000 strain belongs to P. syringae pv. maculicola. This study shows that P. syringae pv. maculicola and P. syringae pv. tomato appear multiclonal, as they did not diverge from a single common ancestral group within the ancestral P. syringae genomospecies 3, and suggests that pathovar specificity within P. syringae may be due to independent genetic events.     

A plant growth-promoting pseudomonad is closely related to the Pseudomonas syringae complex of plant pathogens

Pseudomonas putida GR12-2 is well known as a plant growth-promoting rhizobacterium; however, phylogenetic analysis using the 16S rRNA gene and four housekeeping genes indicated that this strain forms a monophyletic group with the Pseudomonas syringae complex, which is composed of several species of plant pathogens. On the basis of these sequence analyses, we suggest that P. putida GR12-2 be redesignated as P. syringae GR12-2. To compare the ecological roles of P. syringae GR12-2 with its close relatives P. syringae pathovar (pv.) tomato DC3000 and P. syringae pv. syringae B728a, we investigated their ability to cause disease and promote plant growth. When introduced on tobacco or tomato leaves, P. syringae GR12-2 was unable to elicit a hypersensitive response or cause disease, which are characteristic responses of P. syringae DC3000 and B728a, nor were type III secretion system genes required for virulence detected in P. syringae GR12-2 by PCR or DNA hybridization. In contrast to P. syringae GR12-2, neither of the phytopathogens was able to promote root growth when inoculated onto canola seeds. Although commensals and nonpathogens have been reported among the strains of the P. syringae complex, P. syringae GR12-2 is a mutualist and a phytostimulator.     

Pseudomonas syringae pv. coriandricola, Incitant of Bacterial Umbel Blight and Seed Decay of Coriander (Coriandrum sativum L.) in Germany

Since 1987 a devastating disease has occurred in coriander in Germany, characterized by dark-brown discoloration of blossoms and umbels. water-soaked and brown spots on leaves and stems, seed decay and willing. Infected tissue always contained large quantities of Gramnegative, rod-shaped, motile bacteria with few polar flagella. Tests for LOPAT reactions showed the bacteria to be positive for levan-production and tobacco hypersensitivity reaction but negative for oxidase reaction, rot of potato slices and arginine dihydrolase. The bacteria failed to produce fluorescent pigment on King's medium B but revealed a blue fluorescence after growing in a liquid medium without Fe, According to further standard nutritional, biochemical and physiological tests the coriander pathogen belongs to Pseudomonas group la. i.e. Pseudomonas syringae . Also, the fatty acid composition revealed a very close similarity to Pseuodomonas syringae . On Biolog plates the coriander strains showed a uniform metabolic pattern and could clearly be distinguished from other Pseudomonas syringae pathovars.
Typical hosts of Pseudomonas syringae pv. syringae were not infected by the coriander pathogen. Also, most tested umbelliferae species reacted resistant towards the pathogen. Typical disease symptoms, such as persistent water-soaked lesions, were incited only a Coriandrum saticum Animi majus and Levisticum offieinale . The studies revealed that the pathogen described is a separate pathovar of Pseudomonas syringae not included in the approved list of P. syringae pathovars. The name Pseudomonas syringae pv. coriandricola is proposed. Strain GSPB 1965 has been deposited in the NCPPB as pathotype strain (no. 3781).     

Sequence diversity of rulA among natural isolates of Pseudomonas syringae and effect on function of rulAB-mediated UV radiation tolerance

The rulAB locus confers tolerance to UV radiation and is borne on plasmids of the pPT23A family in Pseudomonas syringae. We sequenced 14 rulA alleles from P. syringae strains representing seven pathovars and found sequence differences of 1 to 12% within pathovar syringae, and up to 15% differences between pathovars. Since the sequence variation within rulA was similar to that of P. syringae chromosomal alleles, we hypothesized that rulAB has evolved over a long time period in P. syringae. A phylogenetic analysis of the deduced amino acid sequences of rulA resulted in seven clusters. Strains from the same plant host grouped together in three cases; however, strains from different pathovars grouped together in two cases. In particular, the rulA alleles from P. syringae pv. lachrymans and P. syringae pv. pisi were grouped but were clearly distinct from the other sequenced alleles, suggesting the possibility of a recent interpathovar transfer. We constructed chimeric rulAB expression clones and found that the observed sequence differences resulted in significant differences in UV (wavelength) radiation sensitivity. Our results suggest that specific amino acid changes in RulA could alter UV radiation tolerance and the competitiveness of the P. syringae host in the phyllosphere.     

Angular Leaf Spot of Iron Wood Incited by a Pathovar of Pseudomonas syringae

A fluorescent pseudomonad inciting brown angular leaf spots on iron wood (Parotia persica) in Mazandaran forest was isolated and identified as a pathovar of Pseudomonas syringae. Strains assimilated adonitol and L-tartrate but not lactate or D-tartrate as carbon sources for growth. The electrophoretic profiles of cell proteins of strains isolated from iron wood were very similar but differred markedly from protein profile of P. syringae pv. syringae.     

Plasmid analysis and variation in Pseudomonas syringae

Total plasmid DNA was successfully isolated from 46 of 55 strains of Pseudomonas syringae . Electrophoretic separation after digestion with restriction endonuclease Eco RI gave reproducible banding patterns. Cluster analysis of banding data grouped all strains of pathovar (pv.) pisi separately from pv. glycinea , pv. phaseolicola and pv. syringae . Pathovars glycinea and phaseolicola were more similar to each other than to pv. pisi. A relationship between fragment banding patterns and race structure within pv. pisi was observed.     

Pto- and Prf-mediated recognition of AvrPto and AvrPtoB restricts the ability of diverse pseudomonas syringae pathovars to infect tomato

The molecular basis underlying the ability of pathogens to infect certain plant species and not others is largely unknown. Pseudomonas syringae is a useful model species for investigating this phenomenon because it comprises more than 50 pathovars which have narrow host range specificities. Tomato (Solanum lycopersicum) is a host for P. syringae pv. tomato, the causative agent of bacterial speck disease, but is considered a nonhost for other P. syringae pathovars. Host resistance in tomato to bacterial speck disease is conferred by the Pto protein kinase which acts in concert with the Prf nucleotide-binding lucine-rich repeat protein to recognize P. syringae pv. tomato strains expressing the type III effectors AvrPto or AvrPtoB (HopAB2). The Pto and Prf genes were isolated from the wild tomato species S. pimpinellifolium and functional alleles of both of these genes now are known to exist in many species of tomato and in other Solanaceous species. Here, we extend earlier reports that avrPto and avrPtoB genes are widely distributed among pathovars of P. syringae which are considered nonhost pathogens of tomato. This observation prompted us to examine the possibility that recognition of these type III effectors by Pto or Prf might contribute to the inability of many P. syringae pathovars to infect tomato species. We show that 10 strains from presumed nonhost P. syringae pathovars are able to grow and cause pathovar-unique disease symptoms in tomato leaves lacking Pto or Prf, although they did not reach the population levels or cause symptoms as severe as a control P. syringae pv. tomato strain. Seven of these strains were found to express avrPto or avrPtoB. The AvrPto- and AvrPtoB-expressing strains elicited disease resistance on tomato leaves expressing Pto and Prf. Thus, a gene-for-gene recognition event may contribute to host range restriction of many P. syringae pathovars on tomato species. Furthermore, we conclude that the diverse disease symptoms caused by different Pseudomonas pathogens on their normal plant hosts are due largely to the array of virulence factors expressed by each pathovar and not to specific molecular or morphological attributes of the plant host.