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)     

Closely Related Plasmid Replicons Coexisting in the Phytopathogen Pseudomonas syringae Show a Mosaic Organization of the Replication Region and Altered Incompatibility Behavior

Many Pseudomonas syringae strains contain native plasmids that are important for host-pathogen interactions, and most of them contain several coexisting plasmids (pPT23A-like plasmids) that cross-hybridize to replication sequences from pPT23A, which also carries a gene cluster coding for the phytotoxin coronatine in P. syringae pv. tomato PT23. In this study, three functional pPT23A-like replicons were cloned from P. syringae pv. glycinea race 6, suggesting that the compatibility of highly related replicons is a common feature of P. syringae strains. Hybridization experiments using three separate incompatibility determinants previously identified from pPT23A and the rulAB (UV radiation tolerance) genes showed that the organization of the replication region among pPT23A-like plasmids from several P. syringae pathovars is poorly conserved. The putative repA gene from four pPT23A-like replicons from P. syringae pv. glycinea race 6 was amplified by using specific primers. The restriction profiles of the resulting PCR products for the race 6 plasmids were more similar to each other than they were to that of pPT23A. These data, together with the existence of other cross-hybridizing DNA regions around the replicon among the race 6 pPT23A-like plasmids, suggest that some of these plasmids may have originated from duplication events. Our results also imply that modifications of the repA sequences and the poor conservation of putative maintenance determinants contribute to the suppression of incompatibility among members of the pPT23A-like family, thus enhancing the genomic plasticity of P. syringae.     

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.     

Cloning and Expression of Genes Required for Coronamic Acid (2-Ethyl-1-Aminocyclopropane 1-Carboxylic Acid), an Intermediate in the Biosynthesis of the Phytotoxin Coronatine

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.     

Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences

Strain PT23 of Pseudomonas syringae pv, tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.     

Phylogenetic Analysis of the pPT23A Plasmid Family of Pseudomonas syringae

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.     

Characterization of Plasmids Encoding the Phytotoxin Coronatine in Pseudomonas syringae

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway.     

Plasmid-mediated production of the phytotoxin coronatine in Pseudomonas syringae pv. tomato.

Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were previously generated by using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants either were missing pPT23A (29 mutants) or contained deletions in this plasmid (4 mutants). These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce coronatine. A bioassay for coronatine suggested that PS61(pPT23A) transconjugants were able to make this phytotoxin. In a chemical analysis, organic acids were isolated from PT23.2, PS61, and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The derivatized organic acids extracted from PT23.2 and PS61(pPT23A) contained peaks that corresponded to coronafacic acid, coronafacoylvaline, and coronatine, but these were absent in the extracts from the wild-type strain PS61. The identification of these components was confirmed by combined gas chromatography-mass spectrophotometry. Therefore, the acquisition of pPT23A by PS61 resulted in biosynthesis of coronafacic acid, coronafacoylvaline, and coronatine, clearly demonstrating the involvement of pPT23A in coronatine production in P. syringae pv. tomato.     

Biological and Molecular Detection of Toxic Lipodepsipeptide-Producing Pseudomonas syringae Strains and PCR Identification in Plants

Toxin-based identification procedures are useful for differentiating Pseudomonas syringae pathovars. A biological test on peptone-glucose-NaCl agar in which the yeast Rhodotorula pilimanae was used proved to be more reliable for detecting lipodepsipeptide-producing strains of P. syringae than the more usual test on potato dextrose agar in which Geotrichum candidum is used. A PCR test performed with primers designed to amplify a 1,040-bp fragment in the coding sequence of the syrD gene, which was assumed to be involved in syringomycin and syringopeptin secretion, efficiently detected the gene in pathovars that produce the lipodepsipeptides. Comparable results were obtained in both tests performed with strains of the syringomycin-producing organisms P. syringae pv. syringae, P. syringae pv. atrofaciens, and P. syringae pv. aptata, but the PCR test failed with a syringotoxin-producing Pseudomonas fuscovaginae strain. The specificity of the test was verified by obtaining negative PCR test results for related pathovars or species that do not produce the toxic lipodepsipeptides. P. syringae pv. syringae was detected repeatedly in liquid medium inoculated with diseased vegetative tissue and assayed by the PCR test. Our procedure was also adapted to detect P. syringae pv. morsprunorum with a cfl gene-based PCR test.     

Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids

Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.     

Characterization of pPT23B, the Plasmid Involved in Syringolide Production by Pseudomonas syringae pv. tomato PT23

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.     

Production and Comparison of Peptide Siderophores from Strains of Distantly Related Pathovars of Pseudomonas syringae and Pseudomonas viridiflava LMG 2352

The production of peptide siderophores and the variation in siderophore production among strains of Pseudomonas syringae and Pseudomonas viridiflava were investigated. An antibiose test was used to select a free amino acid-containing agar medium favorable for production of fluorescent siderophores by two P. syringae strains. A culture technique in which both liquid and solid asparagine-containing culture media were used proved to be reproducible and highly effective for inducing production of siderophores in a liquid medium by the fluorescent Pseudomonas strains investigated. Using asparagine as a carbon source appeared to favor siderophore production, and relatively high levels of siderophores were produced when certain amino acids were used as the sole carbon and energy sources. Purified chelated siderophores of strains of P. syringae pv. syringae, P. syringae pv. aptata, P. syringae pv. morsprunorum, P. syringae pv. tomato, and P. viridiflava had the same amino acid composition and spectral characteristics and were indiscriminately used by these strains. In addition, nonfluorescent strains of P. syringae pv. aptata and P. syringae pv. morsprunorum were able to use the siderophores in biological tests. Our results confirmed the proximity of P. syringae and P. viridiflava; siderotyping between pathovars of P. syringae was not possible. We found that the spectral characteristics of the chelated peptide siderophores were different from the spectral characteristics of typical pyoverdins. Our results are discussed in relation to the ecology of the organisms and the conditions encountered on plant surfaces.     

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.     

Plasmid-Determined Copper Resistance in Pseudomonas syringae from Impatiens

A strain of Pseudomonas syringae was recently identified as the cause of a new foliar blight of impatiens. The bacterium was resistant to copper compounds, which are used on a variety of crops for bacterial and fungal disease control. The bacterium contained a single 47-kilobase plasmid (pPSI1) that showed homology to a copper resistance operon previously cloned and characterized from P. syringae pv. tomato plasmid pPT23D (D. Cooksey, Appl. Environ. Microbiol. 53:454-456, 1987). pPSI1 was transformed by electroporation into a copper-sensitive P. syringae strain, and the resulting transformants were copper resistant. A physical map of pPSI1 was constructed, and the extent of homology to pPT23D outside the copper resistance operon was determined in Southern hybridizations. The two plasmids shared approximately 20 kilobases of homologous DNA, with the remainder of each plasmid showing no detectable homology. The homologous regions hybridized strongly, but there was little or no conservation of restriction enzyme recognition sites.     

Construction and Use of a Nonradioactive DNA Hybridization Probe for Detection of Pseudomonas syringae pv. Tomato on Tomato Plants

Pseudomonas syringae pv. tomato, the causal agent for bacterial speck of tomato, produces the phytotoxin coronatine. A 5.3-kilobase XhoI fragment from the chromosomal region controlling toxin production was cloned into the plasmid pGB2, and the resulting recombinant plasmid, pTPR1, was tested for its ability to serve as a diagnostic probe for P. syringae pv. tomato. In a survey of 75 plant-associated bacteria, pTPR1 hybridized exclusively to those strains that produced coronatine. The detection limit for this probe, which was labeled with the Chemiprobe nonradioactive reporter system, was approximately 4 × 10³ CFU of lesion bacteria. During the 1989 growing season, a total of 258 leaf and fruit lesions from nine tomato fields were screened for P. syringae pv. tomato by using pTPR1 and the culture method of detection. The best agreement between the two methods, 90%, occurred early in the season with samples taken from relatively young (5-week-old) plants. Young plants also had a higher percentage of P. syringae pv. tomato-positive lesions. P. syringae pv. tomato was the only coronatine producer recovered from the nine tomato fields. All 244 P. syringae pv. tomato strains isolated during this study reacted strongly with the probe. The P. syringae pv. tomato population of healthy field tomato leaves was determined by a pTPR1 colony hybridization procedure. Every probe-positive colony that was isolated and characterized was identified as P. syringae pv. tomato. The pTPR1 probe should expedite disease diagnosis and facilitate epidemiological studies of this pathogen. It also should aid in screening transplant seedlings for bacterial speck infestation.     

Physical and functional characterization of the gene cluster encoding the polyketide phytotoxin coronatine in Pseudomonas syringae pv. glycinea.

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The coronatine synthesis genes in PG4180 were previously shown to reside on a 90-kb plasmid designated p4180A. In the present study, clones containing a 34-kb region of p4180A were saturated with Tn5, and 71 unique mutations were recombined into p4180A by marker exchange. The effect of each mutation on coronatine synthesis was determined by analyzing the organic acids produced by the mutants by reverse-phase high-performance liquid chromatography. The organic acids of selected mutants were derivatized to their methyl esters and analyzed by gas chromatography and gas chromatography-mass spectrometry. Mutations in a 20.5-kb region of p4180A completely blocked the synthesis of coronafacic acid and coronatine. Mutations within a 4.4-kb region of p4180A prevented the formation of coronatine but allowed for production of coronafacic acid, coronafacoylvaline, coronafacoylisoleucine, and coronafacoylalloisoleucine. The phenotypes of selected mutants were further confirmed in feeding experiments in which coronafacic acid or coronamic acid was added to the culture media. The results of this study allow us to speculate on the likely sequence of steps in the later stages of coronatine biosynthesis.     

Genes required for pathogenicity and hypersensitivity are conserved and interchangeable among pathovars of Pseudomonas syringae

Summary A group of pathogenicity genes was previously identified in Pseudomonas syringae pv. phaseolicola which controls the ability of the pathogen to cause disease on bean and to elicit the hypersensitive response on non-host plants. These genes, designated hrp, are located in a ca. 20 kb region which was referred to as the hrp cluster. Homologous sequences to DNA segments derived from this region were detected in several pathovars of P. syringae but not in symbiotic, saprophytic or other phytopathogenic bacteria. A Tn5-induced Hrp^- mutation was transferred from P. syringae pv. phaseolicola to P. syringae pv. tabaci and to three races of P. syringae pv. glycinea by marker exchange mutagenesis. The resulting progeny were phenotypically Hrp^-, i.e. no longer pathogenic on their respective hosts and unable to elicit the hypersensitive response on non-host plants. These mutants were restored to wild-type phenotype upon introduction of a recombinant plasmid carrying the corresponding wild-type locus from P. syringae pv. phaseolicola. The marker exchange mutants of P. syringae pv. glycinea psg0 and Psg5 which carry different avr genes for race specific avirulence did not elicit a hypersensitive response on incompatible soybean cultivars. It appears, therefore, that P. syringae pathovars possess common genes for pathogenicity which also control their interaction with non-host plants. Furthermore, the expression of race/cultivar specific incompatibility of P. syringae pv. glycinea requires a fully functional hrp region in addition to the avr genes which determine avirulence on single-gene differential cultivars of soybean.     

A gene involved in quinate metabolism is specific to one DNA homology group of Xanthomonas campestris

A gene involved in quinate metabolism was cloned from Xanthomonas campestris pv. juglandis strain C5. The gene, qumA, located on a 4. 2-kb KpnI-EcoRV fragment in plasmid pQM38, conferred quinate metabolic activity to X. c. pv. celebensis. Tn3-spice insertional analyses further located the qumA gene on a region of about 3.0 kb within pQM38. Nucleotide sequencing of this 3.0-kb fragment reveals that the coding region of qumA is 2373 bp, the deduced amino acid sequence of which closely resembles a pyrrolo-quinoline quinone-dependent quinate dehydrogenase of Acinetobacter calcoaceticus. A 0.7 kb SalI-PstI fragment internal to qumA was used as a probe to hybridize against total genomic DNA from 43 pathovars of X. campestris. The fragment hybridized only to total genomic DNA from the four pathovars of DNA homology group 6, X. c. pv. celebensis, X. c. pv. corylina, X. c. pv. juglandis and X. c. pv. pruni, and from X. c. pv. carotae, which belongs to DNA homology group 5. This 0.7 kb fragment was also used as a probe to hybridize BamHI-digested total genomic DNAs from the four pathovars of DNA homology group 6 and X. c. pv. carotae. The restriction fragment length polymorphism pattern of DNA homology group 6 was different from that of X. c. pv. carotae. The probe hybridized to a 5.7-kb BamHI fragment in all four pathovars of group 6 and to a 6.1-kb BamHI fragment in three of four pathovars. It hybridized only to a 9. 9-kb BamHI fragment in X. c. pv. carotae. Quinate metabolism has previously been reported as a phenotypic property specific to X. campestris DNA homology group 6. Accordingly, a combination of the quinate metabolism phenotypic test and Southern hybridization using a qumA-derived probe will be very useful in the identification of pathovars in DNA homology group 6.     

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).     

Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato

Avirulence gene D, cloned from Pseudomonas syringae pv. tomato, caused P. s. pv. glycinea to elicit a hypersensitive defense response on certain cultivars of soybean. Nucleotide sequence data for a 5.6-kb HindIII fragment containing avrD disclosed five long open-reading frames (ORFs) occurring in tandem. The phenotype conferred by avrD was expressed in P. s. pv. glycinea solely by the first of these ORFs (933 bases) that encoded a protein of 34,115 daltons. Neither a signal peptide sequence nor significant regions of hydrophobicity were present that would indicate secretion of the protein or its membrane association. Hybridization analyses revealed that some but not all P. syringae pathovars contained DNA homologous to avrD. This included weak hybridization to all tested races of P. s. pv. glycinea, although none of them express the phenotype conferred by avrD. The avrD gene occurred on an indigenous 75-kb plasmid in several P. s. pv. tomato isolates.