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.     

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

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

Morphology of Leaf Surfaces of Tomato Cultivars in Relation to Possible Invasion into the Leaf by Pseudomonas syringae pv. tomato

The role of morphological characteristics of tomato leaves inthe infection process of the pathogen Pseudomonas syringae pv.tomato was studied in 15 cultivars of varying susceptibility.Natural openings such as stomata, broken trichomes and cuticularcracks were counted. It was found that susceptibility increasedin proportion to the number of these potential penetration sites.However, their role is probably limited because even in thehighly resistant cultivars there are enough natural openingsto enable successful bacterial penetration. Bacterial speck of tomato, Lycopersicon esculentum, phytopathogenic bacteria, plant morphology, Pseudomonas syringae pv. tomato, tomato     

Characterization of a Copper Resistance Plasmid Conserved in Copper-Resistant Strains of Pseudomonas syringae pv. tomato

A 35-kilobase plasmid was conserved among 12 copper-resistant strains of Pseudomonas syringae pv. tomato. Restriction patterns of this plasmid from each strain were identical, and a cloned copper resistance gene from 1 strain hybridized to the same location on the 35-kilobase plasmid of all 12 strains.     

Transmission Electron Microscopy of Susceptible and Resistant Tomato Leaves Following Infection with Pseudomonas syringae pv. tomato

Ultrastructural changes in tomato leaves of susceptible cv. Peto 95 and resistant cv. Ontario 7710 infected with Pseudomonas syringae pv. tomato were followed by transmission electron microscopy. Up to 48 hours from the inoculation host cells of both cultivars looked quite normal and no bacteria were visible in the intercellular spaces; bacterial cells were found only in the substomatal chambers. Afterwards, the leaf cells of cv. Peto 95 began to degenerate and bacteria invaded the intercellular spaces which seemed enlarged. After 15 days the disorganization was complete: tomato cells were plasmolyzed and the intercellular spaces were filled with bacteria. In the leaves of resistant cv. Ontario 7710 no bacteria were observed later than 48 hours and no visible modifications occurred up to 15 days after the inoculation.     

Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant–pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air–liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.     

Response of tomato genotypes to bacterial speck (Pseudomonas syringae pv. tomato)

Two genotypes of tomato A 100 and Ontario 7710 which were inoculated separately with four strains of Pseudomonas syringae pv. tomato differed significantly in disease severity (susceptibility) to bacterial speck. At both concentrations of inoculum of each strain used (10⁷ and 10⁸ cfu/ml) A 100 appeared to be highly susceptible whereas Ontario 7710 showed very low or no susceptibility. The significant differences in virulence between strains and in response of tomato plants in three replicate experiments were found. Generally, concentration of inoculum 10⁷ cfu/ml was too low to induce consistent level of disease severity. The obtained results indicate the importance of consistent and favorable conditions for disease development in screening of tomato resistance to bacterial speck.     

The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.     

Occurrence of Pseudomonas syringae pv. Tomato race 1 in Italy on Pto Gene-Bearing Tomato Plants

Bacterial speck symptoms on leaves of the tomato cultivar Erminia Fl (Petoseed), heterozygous for the Pto resistant gene, were observed in June 1995 in Northern Italy. Using individual-lesion isolation, 8 bacterial isolates were obtained from the affected leaves, which were identified as Pseudomonas syringae pv. tomato by biochemical, physiological, nutritional and pathogenicity tests. When the differential cultivar Ontario 7710, homozygous for the Pto gene, was inoculated with the bacterial isolates, 4 of them provoked the typical bacterial speck symptoms and therefore belonged to race 1. This is the first occurrence of P. syringae pv. tomato race 1 in Italy and the first time this race has been isolated on a Pto gene-bearing tomato cultivar grown in open field.     

Generation and Characterization of Tn5 Insertion Mutations in Pseudomonas syringae pv. tomato

Tn5-induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by mating this plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for Tn5, pGS9. Km^r transconjugants occurred at frequencies ranging from 2 × 10⁻⁷ to 9 × 10⁻⁶; approximately 5.5% of these transconjugants were also Cm^r, indicating the presence of additional pGS9 DNA sequences. Approximately 1% of the Km^r Cm^s mutants were auxotrophic. Southern blot analysis revealed that the Tn5 element had inserted into one unique site on the chromosome for each Km^r Cm^s transconjugant examined. Physical and genetic tests of Tn5-induced auxotrophs showed that Tn5 mutations in P. syringae pv. tomato were very stable and that secondary transposition of Tn5 or its insertion sequence IS50 was a rare event. Nine of 920 Km^r Cm^s transconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each of the virulence mutants was the result of a unique single-site Tn5 insertion. Five mutants also failed to induce a hypersensitivity reaction on tobacco.     

Inheritance and sources of resistance to bacterial speck of tomato caused by Pseudomonas syringae pv. tomato

Inheritance of resistance to bacterial speck of tomato was determined by analysing F₁ F₂ and backcross progenies of crosses involving a susceptible (VF-198) and a resistant cultivar (Rehovot-13). The results fit the hypothesis that resistance is controlled by a single dominant gene in interaction with minor genes. Cultivar susceptibility to Pseudomonas syringae pv. tomato was tested under greenhouse conditions under high inoculum pressure using infested tomato seeds together with infested soils and spray-inoculated wounded plants. Of 21 species, cultivars and lines, Rehovot-13, Ontario 7710 and Lycopersiconpimpinellifolium P.I. 126927 were found to be resistant to the pathogen. VF-198 and Tropic-VF were the most susceptible. Extra Marmande, Saladette, Acc.339944–3 and the wild type Lycopersicon esculentum var. cerasiforme were moderately resistant.     

The 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 VI. Serogroup III includes the P. syringae pv. atrofaciens strains that were isolated from cereals in the United Kingdom but not in Ukraine.     

Crystal structure of uroporphyrinogen III synthase from Pseudomonas syringae pv. tomato DC3000

Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole compounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III (uro’gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5 Å resolution by the single wavelength anomalous dispersion (SAD) method. Each psU3S molecule consists of two domains interlinked by a two-stranded antiparallel β-sheet. The conformation of psU3S is different from its homologous proteins because of the flexibility of the linker between the two domains, which might be related to this enzyme’s catalytic properties. Based on mutation and activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S.     

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.     

Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

Differential effects of flagellins from Pseudomonas syringae pv. tabaci,tomato and glycinea on plant defense response

To investigate the factor that determines incompatible interactions between Pseudomonas syringae pv. tabaci and non-host plants, an elicitor of hypersensitive reaction (HR) was partially purified from the supernatant of a nutrient-poor medium of bacterial culture by DEAE column chromatography. The major protein in the elicitor-active fractions was identified as a flagellin which is a component of flagellar filaments. The flagellins purified from P. syringae pv. tomato and glycinea, incompatible pathogens of tobacco plants, induced fragmentation of chromosomal DNA and oxidative burst accompanied by programmed cell death in tobacco (Nicotiana tabacum) Bright Yellow (BY-2) cells, but the flagellin from pv. tabaci, a compatible pathogen, did not. However, the amino acid sequences of flagellins deduced from fliC genes showed a high homology among these P. syringae pathovars. In particular, the amino acid sequences of pv. tabaci and glycinea are completely identical. However, both recombinant flagellins produced in Escherichia coli possess HR-inducing activity in BY-2 cells. These results indicate that the post-translational modification of flagellins has an important role for HR-inducing ability in tobacco cells. Furthermore, we discuss the cause of a different elicitor activity among flagellins on tobacco cells and the role of flagellins in the determining specificity.     

Assessment of strains of Pseudomonas syringae pv. tomato from Tanzania for resistance to copper and streptomycin

Abstract

Fifty-six strains of Pseudomonas syringae pv. tomato (P.s. pv. tomato) were collected from tomato-producing areas in Tanzania and assessed for resistance to copper and antibiotics. The collection was done from three tomato-producing regions (Morogoro, Arusha and Iringa), representing three different ecological conditions in the country. After isolation and identification, the P. s. pv. tomato strains were grown on King's medium B (KB) amended with 20% copper sulphate (w/v). The strains were also assessed for resistance to antibiotics. Results indicated that there was widespread resistance of the P. s. pv. tomato strains to copper sulphate. The highest level of resistance was recorded from the Arusha region (Northern Tanzania), 83.3% of the P. s. pv. tomato strains from that region showed resistance to copper sulphate. This was followed by Iringa region (Southern Tanzania), from where strains of the pathogen were moderately resistant to copper sulphate, such that 54.0% of them were able to grow on the KB medium amended with 20% (w/v) of the copper compound.

Out of seven strains of P. s. pv. tomato from Morogoro region (Central Tanzania) included in the study, five (71.5%) were resistant to copper sulphate. The only strain of P. s. pv. tomato from the Dodoma region (Central Tanzania, but with a different ecological condition from the Morogoro region) included in the study was unable to grow on the medium containing 20% copper sulphate. None of the P. s. pv. tomato strains in the four regions included in the study were resistant to streptomycin sulphate. These results suggest that in the Arusha and Iringa regions of Tanzania, there might be possibilities of excessive use of copper compounds in tomato production, such that strains of P. s. pv. tomato strains in the areas have become resistant to the compounds.