Genetic Characterization of Pseudomonas syringae pv. syringae Strains from Stone Fruits in California

Strains of Pseudomonas syringae pv. syringae were isolated from healthy and diseased stone fruit tissues sampled from 43 orchard sites in California in 1995 and 1996. These strains, together with P. syringae strains from other hosts and pathovars, were tested for pathogenicity and the presence of the syrB and syrC genes and were genetically characterized by using enterobacterial repetitive intergenic consensus (ERIC) primers and PCR. All 89 strains of P. syringae pv. syringae tested were moderately to highly pathogenic on Lovell peach seedlings regardless of the host of origin, while strains of other pathovars exhibited low or no pathogenicity. The 19 strains of P. syringae pv. syringae examined by restriction fragment length polymorphism analysis contained the syrB and syrC genes, whereas no hybridization occurred with 4 strains of other P. syringae pathovars. The P. syringae pv. syringae strains from stone fruit, except for a strain from New Zealand, generated ERIC genomic fingerprints which shared four fragments of similar mobility. Of the P. syringae pv. syringae strains tested from other hosts, only strains from rose, kiwi, and pear generated genomic fingerprints that had the same four fragments as the stone fruit strains. Analysis of the ERIC fingerprints from P. syringae pv. syringae strains showed that the strains isolated from stone fruits formed a distinct cluster separate from most of the strains isolated from other hosts. These results provide evidence of host specialization within the diverse pathovar P. syringae pv. syringae.     

Bacterial canker of stone-fruits: Infection experiments with Pseudomonas mors-prunorum and P. syringae

The effect was compared of inoculating Pseudomonas mors-prunorum and P. syringae at high inoculum concentration through wounds in plum stems and cherry branches, and through cherry-leaf scars. In the wound inoculations a North American cherry strain of P. syringae was considerably more virulent than any of three indigenous cherry strains of P. mors-prunorum, but two pear strains of P. syringae were less virulent. P. mors-prunorum showed a much greater capacity to invade through leaf scars, particularly towards the end of the leaf-fall period, when all three P. syringae strains were largely ineffective. Evidence is discussed which suggests that the greater infectivity of the P. mors-prunorum strains through leaf scars was related to their capacity to colonize the host tissues from small inocula. The P. syringae strains died out in cherry cankers earlier than P. mors-prunorum strains and they were less stable during host passage. The results of the experiments indicate that virulence in these organisms is a complex phenomenon and determined by several factors.     

Induced Resistance in Tomato Against Corynebacterium michiganense pv. michiganense by Prior Inoculation with Pseudomonas syringae Pathovars

Prior inoculation of wounded tomato petioles with a minimum concentration of 5 × 10⁴ cells per wound of various Pseudomonas syringae pathovars completely protected plants against subsequent infection with Corynebacterium michiganense pv. michiganense inoculated on the same site. Only living cells induced effective protection. In protected tissue, cells of Corynebacterium michiganense pv. michiganense remained localized at the inoculation site and their multiplication was restricted. Irrespective of the cell number introduced, initial population decreased slowly and then levelled off below the initial inoculum level. This level remained constant throughout the experimental period (15 days). Similarly, the, cell number of the inducer Pseudomonas syringae pv. phaseolicola levelled off at ca. 10⁶ cells per plant. The protection was not systemic and could be eliminated by removing the upper 5 mm of the inoculated wound tissues containing, the inducer.     

Effectiveness of alkyl dimethyl benzyl ammonium chloride in reducing the population of Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. syringae in tomatoes,beans, and peppers

Abstract

Microbial contamination of fruits and vegetables during growth, processing, and post-harvest is a serious problem in agricultural sectors. A study was undertaken to investigate the efficacy of alkyl dimethyl benzyl ammonium chloride (ADBAC) in reducing the population of Xanthomonas campestris pv. vesicatoria, and Pseudomonas syringae pv. syringae on tomatoes, beans, and peppers. Tomatoes, beans, and peppers were inoculated by dipping in bacteria for 15 min then fruits were dried for 2 hour at ambient temperature before they were treated with 0.1, 1, 10, 100, and 1000 ppm of ADBAC. Treatments with 10, 100, and 1000 ppm ADBAC caused an 8-log CFU/ml reduction of X. campestris pv. vesicatoria on surfaces of tomatoes. Treatments with 100 and 1000 ppm ADBAC caused an 8-log CFU/ml reduction of P. syringae pv. syringae and X. campestris pv. vesicatoria on surfaces of tomatoes and peppers, respectively. However, treatment of surfaces of beans with 1000 ppm of ADBAC caused an 8-log CFU/ml reduction of P. syringae pv. syringae. Overall, a 50% reduction on population counts of both pathogens was achieved with 100 and 1000 ppm ADBAC. No X. campestris pv vesicatoria, P. syringae pv. syringae, or other bacteria were detected on the control fruits inoculated with sterile distilled water. This study's findings suggest that ADBAC has good bactericidal and sanitizing activities and could potentially be useful as a new sanitizer for food safety.     

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.     

First Report of Bacterial Leaf Spot (Pseudomonas syringae pv. coriandricola) of Coriander in Spain

Bacterial leaf spot symptoms of coriander were first observed in January 2003 in three coriander fields in the valley region of the Axarquía (Málaga, Spain), showing a very high incidence. Pseudomonas syringae pv. coriandricola was consistently isolated from diseased plants, identified and its pathogenicity on coriander could be proved. The effective inoculum dose (ED₅₀) of the isolated strains was estimated and it was very similar to those displayed by the P. syringae pv. coriandricola reference strains used as control. This is the first report of bacterial leaf spot on coriander in Spain.     

The Occurrence of Bacterial Red Streak of Sugarcane Caused by Pseudomonas syringae pv. syringae in Iran

A bacterial leaf streak disease characterized by reddish, narrow (1–2 mm wide) streaks of variable size, and occasionally with bleached centers, was found in sugarcane (Saccharum, interspecific hybrid) fields in northern Iran. The incitant bacterium was identified as Pseudomonas syringae pv. syringae (P. s. syringae). The disease is similar in aetiology to the sugarcane ‘red streak’ disease reported recently from Japan. Cultivardependent variations in symptoms were noted., Difference in pathogenicity as well as in electrophoretic profile of cell proteins between strains of P.s. syringae causing red streak in sugarcane and those causing canker on stone fruit trees, were observed.     

Cloning of genes required for hypersensitivity and pathogenicity inPseudomonas syringae pv.aptata

A genomic library ofPseudomonas syringae pv.aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kbEcoRI fragment of the cosmid pHIR11, containing thehrp (hypersensitiveresponse andpathogenicity) gene cluster of the closely related bacteriumPseudomonas syringae pv.syringae strain 61, was used as a probe to identify a homologoushrp gene cluster inP. syringae pv.aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium,Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis ofEcoRI-digested genomic DNA ofP. syringae pv.aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome ofP. syringae pv.aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kbBglII fragment of pHIR11. These results indicate thatP. syringae pv.aptata harbourshrp genes that are similar to, but arranged differently from, homologoushrp genes ofP. syringae pv.syringae.Abbreviations HR hypersensitive response - Hrp mutant unable to induce HR and pathogenicity - Psa Pseudomonas syringae pv.aptata - Pss Pseudomonas syringae pv.syringae - Ea Erwinia amylovora     

Fatty Acid Composition of Pseudomonas syringae pv. savastanoi

Over 85% of total cellular fatty acids of 30 strains of P. syringae pv. savastanoi, grown for one day at 28 °C on King's medium B (KB) agar, were 12:0 (5.0%), 16:0 (27.5%), 16:1 (36.7%) and 18:1 (16.8%). Three hydroxy-substituted fatty acids comprised 7.2% of the total and 22 other minor components, each occurring at concentrations of less than 1%, comprised an additional 4%. Three percent were unidentified components. Cells grown for 3 and 6 days on KB agar contained lower concentrations of the unsaturated 16:1 (30.4 and 21.1%, respectively), and higher concentrations of branched-chain and cyclopropane fatty acids than one-day old cells. No consistent differences in fatty acid composition could be detected between virulent and avirulent strains, nor between pv. savastanoi and other pathovars of P. syringae. However, when cells were grown on a chemically-defined medium for 6 days, concentrations of 16:0 and a tentatively-identified 17-carbon hydroxy fatty acid were higher, and those of 12:0 and 16:1 were lower in strains from Fraxinus than from Olea. P. fluorescens (7 strains) and P. viridiflava (6 strains) could be differentiated from each other but not from P. syringae.     

Scanning Electron Microscopy of Invasion of Apple Leaves and Blossoms by Pseudomonas syringae pv. syringae

Scanning electron microscopy indicated that Pseudomonas syringae pv. syringae L795 entered leaves through stomata and multiplied in the substomatal chambers. Strain L195 applied to blossoms colonized stigmas and also occurred in intercellular spaces of styles. Nonpathogenic strain L796 failed to colonize blossoms. This study suggests that inoculum of pathogenic P. syringae pv. syringae builds up on apple leaves and blossoms.     

Phage biocontrol to combat Pseudomonas syringae pathogens causing disease in cherry

Bacterial canker is a major disease of Prunus species, such as cherry (Prunus avium). It is caused by Pseudomonas syringae pathovars, including P. syringae pv. syringae (Pss) and P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2). Concerns over the environmental impact of, and the development of bacterial resistance to, traditional copper controls calls for new approaches to disease management. Bacteriophage-based biocontrol could provide a sustainable and natural alternative approach to combat bacterial pathogens. Therefore, seventy phages were isolated from soil, leaf and bark of cherry trees in six locations in the south east of England. Subsequently, their host range was assessed against strains of Pss, Psm1 and Psm2. While these phages lysed different Pss, Psm and some other P. syringae pathovar isolates, they did not infect beneficial bacteria such as Pseudomonas fluorescens. A subset of thirteen phages were further characterized by genome sequencing, revealing five distinct clades in which the phages could be clustered. No known toxins or lysogeny-associated genes could be identified. Using bioassays, selected phages could effectively reduce disease progression in vivo, both individually and in cocktails, reinforcing their potential as biocontrol agents in agriculture.     

Characterization and genetic diversity of Pseudomonas syringae isolates from stone fruits in north‐western Iran

From 33 Iranian fluorescent Pseudomonas isolates originating from symptomatic tissues of peach (Prunus persica), plum (Prunus domestica), sweet (Prunus avium) and sour cherry (Prunus cerasus), 27 were identified as Pseudomonas syringae using LOPAT tests. Further characterization of those isolates by GATTa and L‐lactate utilization tests and the detection of syringomycin and coronatine and yersiniabactin coding genes showed that five of them belonged to race 1 and four to race 2 of P. syringae pv. morsprunorum (Psm) and eighteen other isolates were identified as P. syringae pv. syringae (Pss). Based on the analysis of the fingerprint patterns generated by REP, ERIC and BOX‐PCR, the strains were differentiated into three main groups at the 67% similarity level. Strains of the groups 1, 2 and 3 belong to Psm race 1, Psm race 2 and Pss, respectively. Rep‐PCR analysis showed high intra‐pathovar variation within the Pss isolates, which grouped into four distinct clusters. Using the REP primers, the percentage of polymorphic loci was 74.61%, whereas with BOX and ERIC primers, it was 60.5 and 55.21%, respectively. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 1 and 2 strains from stone fruit trees in Iran.     

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.     

Cell Surface Properties of Pseudomonas syringae pv. phaseolicola Wild-type and hrp Mutants

The cell surface hydrophobicity and charge as well as surface polysaccharides of eight independent prototrophic hrp::-Tn5 mutants (Lindgren et al., J. Bacteriol. 168 , 512–522, 1986) were compared to the wild-type parent strain NPS3121 of Pseudomonas syringae pv. phaseolicola. No significant differences were found in cell surface charge, but mutant strain NPS4005 exhibited significantly lower cell surface hydrophobicity than the wild-type and the other mutant strains. The mutant strains all retained the ability to produce the exopolysaccharides (EPS) levan, a neutral fructan, and alginate, an acidic polymer. Relative amounts of EPS produced in vitro was dependent on culture conditions. Lipopolysaccharide (LPS) chemotypes were similar for all nine strains. Chemical as well as ¹³C-NMR analyses of the O-antigens from four wild-type strains of P. s. pv. phaseolicola representing two physiological races as well as the O-antigens of two strains of P. s. pv. syringae which belong to the same serogroup as P. s. pv. phaseolicola indicated that all of the O-antigens were very similar if not identical. LPS of three strains of P. s. pv. phaseolicola produced in vitro or in planta were also compared and no significant differences were detected. The altered phenotype of the Tn5 mutants of P. s. pv. phaseolicola does not appear to be due to changes in the ability to produce exopolysaccharides or to an altered composition of cell surface polysaccharides (LPS and EPS). However, a change in an unidentified cell surface component(s) leading to lowered cell surface hydrophobicity of mutant strain NPS4005 may be important.     

Systemic Invasion of Immature Sweet Cherry Fruit by Pseudomonas syringae pv. morsprunorum Through Blossoms1

Cherry blossoms inoculated with a rifampicin-resistant strain of Pseudomonas syringae pv. morsprunorum died or gave rise to fruits containing necrotic spots at or near the blossom ends. Scanning electron microscopy of developing fruits indicated that the pathogen had invaded the entire pericarp, including the endocarp. Bacteria also spread to the fruit stalk and, to a lesser extent, to the spurs. Mesocarp cells below the lesion collapsed. Infected fruit, stalks, and spurs contained, respectively, ca. 10⁹, 10⁷, and 10² colony forming units of P. syringae pv. morsprunorum as determined by a dilution plate method on an agar medium supplemented with 50 μg/ml rifampicin. This is the first report of systemic spread of P. syringae from blossoms to developing fruit of a deciduous crop.     

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.     

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.     

Identification and expression of the Pseudomonas syringae pv. aptata hrpZPsa gene which encodes an harpin elicitor

A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv. syringae hrpZ gene was identified in Pseudomonas syringae pv. aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants, and in E. coli DH10B (pCCP1069) containing the P. syringae pv. aptata hrp gene cluster. PCR with oligonucleotides, based on the hrpZ_Pss gene and used as primers with the total genomic DNA of P. syringae pv. aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions. The amplicon was cloned into the pGEM-T® plasmid vector, amplified in E. coli DH5 and sequenced. The sequence showed 95%, 83% and 61% identity with those of hrpZ_Pss, hrpZ_Psg and hrpZ_Pst genes encoding the harpins of the P. syringae pv. syringae, glycinea and tomato, respectively. The amplicon was cloned into the pMAL® expression system. The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography. On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P. syringae pv. aptata harpin.     

Bioinformatics Analysis of the Complete Genome Sequence of the Mango Tree Pathogen Pseudomonas syringae pv. syringae UMAF0158 Reveals Traits Relevant to Virulence and Epiphytic Lifestyle

The genome sequence of more than 100 Pseudomonas syringae strains has been sequenced to date; however only few of them have been fully assembled, including P. syringae pv. syringae B728a. Different strains of pv. syringae cause different diseases and have different host specificities; so, UMAF0158 is a P. syringae pv. syringae strain related to B728a but instead of being a bean pathogen it causes apical necrosis of mango trees, and the two strains belong to different phylotypes of pv.syringae and clades of P. syringae. In this study we report the complete sequence and annotation of P. syringae pv. syringae UMAF0158 chromosome and plasmid pPSS158. A comparative analysis with the available sequenced genomes of other 25 P. syringae strains, both closed (the reference genomes DC3000, 1448A and B728a) and draft genomes was performed. The 5.8 Mb UMAF0158 chromosome has 59.3% GC content and comprises 5017 predicted protein-coding genes. Bioinformatics analysis revealed the presence of genes potentially implicated in the virulence and epiphytic fitness of this strain. We identified several genetic features, which are absent in B728a, that may explain the ability of UMAF0158 to colonize and infect mango trees: the mangotoxin biosynthetic operon mbo, a gene cluster for cellulose production, two different type III and two type VI secretion systems, and a particular T3SS effector repertoire. A mutant strain defective in the rhizobial-like T3SS Rhc showed no differences compared to wild-type during its interaction with host and non-host plants and worms. Here we report the first complete sequence of the chromosome of a pv. syringae strain pathogenic to a woody plant host. Our data also shed light on the genetic factors that possibly determine the pathogenic and epiphytic lifestyle of UMAF0158. This work provides the basis for further analysis on specific mechanisms that enable this strain to infect woody plants and for the functional analysis of host specificity in the P. syringae complex.     

Host specificity,pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.