Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance

Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).     

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.     

Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato.

A cosmid library of copper-resistant (Cur) Pseudomonas syringae pv. tomato PT23 plasmid DNA was constructed and mobilized into the copper-sensitive recipient P. syringae pv. syringae PS61. One resultant cosmid clone, pCOP1 (46 kilobases), conferred copper resistance. The PT23 Cur gene(s) was located on pCOP1 by subcloning PstI restriction endonuclease fragments of pCOP1 in the broad-host-range vector pRK404. A subclone containing a 4.4-kilobase PstI fragment conferred Cur on PS61. The Cur gene(s) was further located by insertional inactivation with Tn5. A subcloned fragment internal to the Cur determinant on pCOP2 was probed to plasmid and chromosomal DNA of four copper-resistant and three copper-sensitive strains of P. syringae pv. tomato. The probe hybridized to plasmids in resistant strains, but showed no detectable homology to copper-sensitive strains.     

62-kb Plasmids Harboring <Emphasis Type="Italic">rulAB</Emphasis> Homologues Confer UV-tolerance and Epiphytic Fitness to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> Mango Isolates

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.     

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.     

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.     

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.     

Preliminary description of biocidal (syringomycin) activity in fluorescent plant pathogenic Pseudomonas species

Strains representing the fluorescent plant pathogenic Pseudomonas spp., Ps. agarici , Ps. asplenii , Ps. avellanae , Ps. beteli , Ps. caricapapayae , Ps. cichorii , Ps. corrugata , Ps. ficuserectae , Ps. flectens , Ps. fuscovaginae , Ps. marginalis , Ps. meliae , Ps. savastanoi , Ps. syringae , Ps. tolaasii and Ps. viridiflava were tested for biocidal activity using Aspergillus niger as assay organism. Inhibitory behaviour was found in strains of Ps. asplenii , Ps. blatchfordae , Ps. cichorii , Ps. corrugata , Ps. fuscovaginae , Ps. marginalis , Ps. marginalis pv. pastinacea , Ps. syringae pv. syringae , Ps. syringae pv. aptata , Ps. syringae pv. atrofaciens , Ps. syringae pv. lapsa , Ps. tolaasii , and strains of a Pseudomonas sp. pathogenic to Actinidia , in the Ps. savastanoi genomic sp. Antifungal activity could be identified with the production of members of the syringomycin family of toxins by strains in Ps. syringae , Ps. asplenii and Ps. fuscovaginae . These toxin reactions support suggestions made elsewhere of the synonymy of the latter two species. In a preliminary characterization using tests for stability to heat, protease, acid and alkaline treatments, unknown toxins consistent with syringomycin-like toxins the strains from Actinidia speciesColour RGB 0,0,128. The toxins from Ps. cichorii and from Ps. corrugata differed in their reactions from all other agents. Pseudomonas tolaasii produces the antifungal compound tolaasin. The white line reaction with ' Ps. reactans ', a test for tolaasin production by strains of Ps. tolaasii , was confirmed as specific for this compound. Some of these low molecular weight toxins may be produced by some of these plant pathogenic strains.     

Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants

The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv. actinidiae, P. syringae pv. syringae, and P. marginalis, isolated from kiwifruit plants in Korea and Japan. PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant. Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR. No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA. Nucleotide sequence analysis indicated that the strA sequence of P. syringae pv. actinidiae contained a single nucleotide alteration at position 593 (CAA-->CGA), as compared to Tn5393a in P. syringae pv. syringae. This resulted in an amino acid change, from Gln to Arg.     

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.     

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic and endophytic colonization of leaves

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.     

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.     

Spatial organization of dual-species bacterial aggregates on leaf surfaces

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% +/- 8.2%) than that in monospecific aggregates of these two strains (1.6% +/- 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.     

DNA markers for identification of Pseudomonas syringae pv. actinidiae

The specific DNA fragment was screened by RAPD analysis of Pseudomonas syringae pv. actinidiae, as well as similar strains that were isolated from kiwifruits. The primer C24 detected a fragment that is specific in P. syringae pv. actinidiae. This fragment was cloned. The pathovar-specific fragment was detected from a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae using the cloned fragment as a probe. The sequence size of the cloned fragment was determined as 675 bp. A DNA Database search suggested that the fragment was a novel one. Approximately 9 kb of a single fragment was detected only in the P. syringae pv. actinidiae by a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae. Similar strains were also detected with the use of the cloned fragment as a probe. Since the genomic DNAs were digested with HindIII without a cleavage site, the result reveals that the cloned fragment exists on the genome of P. syringae pv. actinidiae as a single copy. A pair of primers that produced a 492 bp single fragment (only in the strains of P. syringae pv. actinidiae) were synthesized, based on the pathovar-specific sequences of the cloned fragment of P. syringae pv. actinidiae. The development of the primers and probe made it possible to diagnose the bacterial canker infection from leaves or trunks of kiwifruit trees before any symptom appeared on the tree.     

Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci.

Pseudomonas syringae pv. tabaci BR2 produces tabtoxin and causes wildfire disease on tobacco and bean plants. Approximately 2,700 Tn5 insertion mutants of a plasmid-free strain, PTBR 2.024, were generated by using suicide plasmid pGS9. Of these Tn5 mutants, 8 were no longer pathogenic on tobacco plants and 10 showed reduced symptoms. All of the eight nonpathogenic mutants caused typical wildfire disease symptoms on bean plants. Two of the nonpathogenic mutants failed to produce tabtoxin. The eight nonpathogenic mutants have Tn5 insertions into different EcoRI and SalI restriction fragments. The EcoRI fragments containing Tn5 from the eight nonpathogenic mutants were cloned into vector pTZ18R or pLAFR3. A genomic library of the parent strain was constructed in the broad-host-range cosmid pLAFR3. Three different cosmid clones that hybridized to the cloned Tn5-containing fragment from one of the nonpathogenic mutants, PTBR 4.000, were isolated from the genomic library. These clones contained six contiguous EcoRI fragments (a total of 57 kilobases [kb]). A 7.2-kb EcoRI fragment common to all three restored pathogenicity to mutant PTBR 4.000. None of the six EcoRI fragments hybridized to Tn5-containing fragments from the other seven mutants. The 7.2-kb fragment was conserved in P. syringae pv. tabaci and P. syringae pv. angulata, but not in other pathovars or strains. Because the mutants retained pathogenicity on bean plants and because of the conservation of the 7.2-kb EcoRI fragment only in pathovars of tobacco, we suggest that genes on the fragment might be related to host specificity.     

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.     

Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins

Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains.     

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.