Inhibitory effect of Thymus vulgaris and Origanum vulgare essential oils on virulence factors of phytopathogenic Pseudomonas syringae strains

• Pseudomonas syringae is a phytopathogenic bacterium that causes lesions in leaves during the colonisation process. The damage is associated with production of many virulence factors, such as biofilm and phytotoxins. The essential oils of Thymus vulgaris (thyme) and Origanum vulgare (oregano) have been demonstrated to inhibit P. syringae. The aim of this study was to investigate the effects of T. vulgaris and O. vulgare essential oils on production of virulence factors of phytopathogenic P. syringae strains, including anti‐biofilm and anti‐toxins activities.
• The broth microdilution method was used for determination of MIC and biofilm inhibition assays. Coronatine, syringomycin and tabtoxin were pheno‐ and genotypically evaluated.
• Both oils showed good inhibitory activity against P. syringae, with MIC values from 1.43 to 11.5 mg·ml^?1 for thyme and 5.8 to 11.6 mg·ml^?1 for oregano. Biofilm formation, production of coronatine, syringomycin and tabtoxin were inhibited by thyme and oregano essential oil in most strains.
• The results presented here are promising, demonstrating the bactericidal activity and reduction of virulence factor production after treatment with thyme and oregano oil, providing insight into how they exert their antibacterial activity. These natural products could be considered in the future for the control of diseases caused by P. syringae.

     

Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.     

Transfer and stability of drug resistance plasmids inEscherichia coli K12

Mating experiments between pairs of strains ofEscherichia coli containing either the compatible plasmids TP120 (Inc N) and R1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids TP120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and TP113, transfer was only observed in continuous-flow culture systems. Strains ofE. coli containing aggregates of plasmids TP120 and R1 were shown to be stable over 5 months continuous cultivation under carbon limited conditions at a growth rate of 0.1 hours^–1 in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels of resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains ofE. coli initially containing both plasmid TP120 and plasmid R1.     

Toxins of Pseudomonas syringae pv. syringae affect H+-transport across the plasma membrane of maize

The activity of some phytotoxic metabolites of Pseudomonas syringae pv. syringae Van Hall strains B359 and B301 on in vivo and in vitro systems of H⁺-transport across the plasma membrane of maize (Zea mays L., hybrid Paolo) was investigated. In particular syringomycin, the first lipodepsinonapeptide isolated from Pss and already studied in plants and yeasts for its effects on several physiological systems, was compared with the recently described lipodepsipeptides with 22 or 25 amino acid residues, so called syringopeptins. The in vivo activity of the phytotoxins was tested on fusicoccin-stimulated H^?-extrusion from cuttings of maize roots, which was inhibited by both types of toxins, with syringomycin more efficient than the syringopeptins. In vitro the H⁺-ATPase activity of predominantly right-side-out plasma membrane vesicles purified by two-phase partitioning was stimulated by 10 μM syringomycin and inhibited by higher levels, in agreement with the results of others with preparations of dicotyledons. Also the inhibition of the phosphohydrolytic activity of inside-out vesicles of mung bean plasma membrane was confirmed for maize. In both types of vesicles the syringopeptirts were better inhibitors than syringomycin. The pH gradient formed on addition of ATP to predominantly (25% latency) inside-out vesicles was immediately and completely collapsed by syringomycin and syringopeptins; H⁺-pumping was prevented if the toxins were added before ATP. The inhibition was concentration dependent, but at very low concentrations the effect was inverted. The results of the present investigation, carried out with maize preparations, confirm and extend the evidence so far obtained with dicotyledons in favour of the plasma membrane as an important site of interaction of syringomycin with the plant cell. They also indicate that, except for some details, the effects of syringopeptins at the level of the plasma membrane are the same as those of syringomycin.     

The improvement of resistance to bacterial speck in transgenic tomato plants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> mediated transformation

The pto gene, responsible for resistance to Pseudomonas syringae pv. tomato, was transferred to tomato genotype Urfa-2 by the LBA4404 strain of A. tumefaciens harboring the plasmid pPTC8. The presence of nptII and pto genes in transgenic plants was proved by PCR analysis. Insertion of the pto gene into the genome of transgenic plants and expression of the gene were confirmed by southern and northern hybridizations, respectively. The pathogen P. syringae pv. tomato was applied to all leaves of transgenic and control plants. While typical bacterial speck symptoms developed on the leaves of control plants, the transgenic plants did not display any typical symptoms of bacterial speck upon inoculation with strains 1 and 0. Some of these transgenic plants had thicker leaves than the control plants and produced abnormal flowers. The pollen of transgenic plants was used for crossing with control plants to produce F1 transgenic lines. Fruits from crossed transgenic and control plants were obtained, and F1 seeds germinated on Murashige and Skoog medium in the presence of kanamycin have developed F1 seedlings. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 102–110. The text was submitted by the authors in English.     

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.     

In vitro Antibiosis by Pseudomonas syringae Pss22d,Acting Against the Bacterial Blight Pathogen of Soybean Plants,Does Not Influence In planta Biocontrol

The epiphyte Pseudomonas syringae pv. syringae 22d / 93 (Pss22d), isolated from soybean leaves, had been characterized as a promising and species‐specific biocontrol strain in vitro and in planta against the plant pathogen P. syringae pv. glycinea (Psg), which causes bacterial blight of soybean. Three toxins are known to be produced by Pss22d: syringomycin, syringopeptin and 3‐methylarginine (MeArg). In contrast to syringopeptin and syringomycin, MeArg inhibited the growth of Psg in vitro. To examine if the toxins produced by Pss22d are responsible for antagonistic effects in planta, the pathogen Psg was co‐inoculated with either Pss22d wild‐type, a syringopeptin/syringomycin‐negative double mutant (Pss22d.ΔsypA/syrE), or a MeArg‐negative mutant (Pss22d.1) into wounds of pin‐pricked leaves of greenhouse‐grown soybean plants, respectively. In all three cases, the wild‐type Pss22d and its toxin‐deficient mutants prevented development of disease symptoms normally caused by Psg. These results indicated that neither syringopeptin, nor syringomycin, nor MeArg was required for Pss22d’s antagonistic activity in planta. Consequently, factors other than the three toxins may contribute to the intra‐species antagonism in planta.     

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.     

Stability of the recombinant plasmid carrying theBacillus amyloliquefaciens α-amylase gene inB. subtilis

Summary Theα-amylase gene ofBacillus amyloliquefaciens has previously been cloned into pUB110 to give the recombinant plasmid, pKTH10 (Palva 1982. Gene 19:81–87). Strains transformed by this plasmid are promising candidates for industrialα-amylase production. The stability of pKTH10 was determined in variousB. subtilis strains possessing specific alleles which affect the level ofα-amylase secretion.B. subtilis strains carrying pKTH10 were cultivated in starch-containing medium for up to 50 generations without antibiotic selection and then screened for the presence of pKTH10. The plasmid proved stable enough (< 1.0% cured after 50 generations) for industrial batchwise enzyme production in two strains, but in asacU9 strain (thesacU9 mutation increases concominantly the production ofα-amylase levansucrase and proteases) 99.9% of cells had lost pKTH10 after 50 generations, although the parental plasmid (pUB110) was stable in this strain (0.09% cured after 50 generations). The instability of pKTH10 in thesacU9 strain seems somehow to be related to high expression of the clonedα-amylase gene: when grown in a medium restrictingα-amylase production, only 0.53% ofsacU9 cells had lost pKTH10 after 50 generations.     

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.     

Occurrence of heterogeneous R-plasmids during two concurrent epidemics due to multidrug-resistantSalmonella oranienburg

During two different epidemics that started in August–September, 1980, 140 and 6 multidrug-resistant strains ofSalmonella oranienburg were isolated from a Children's Hospital in New Delhi (epidemic 1) and H.N. Das Hospital in Bombay (epidemic 2) respectively. Out of 140, 123 strains showed high levels of resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. All six strains of epidemic 2 and two strains of epidemic 1 were also found resistant to chloramphenicol in addition to the above antibiotics. The genetic characterization of conjugative R-plasmids harbored by these strains revealed that, while strains of epidemic 1 carry a fi^–, 96 megadalton, an unclassified plasmid, the strains isolated during epidemic 2 contain a fi^–, 62 megadalton plasmid of incompatibility group I₁. All the six strains of epidemic 2 were found to produce bacteriocin of Col Ib group. Plasmid transfer studies revealed that the genes for antibiotic resistance and bacteriocin production were coded on a single plasmid of 62 megadalton. The data show the significance of detailed genetic analysis while dealing with the clonal outbreaks due to same resistant serotypes.     

Monoclonal antibodies againstAgrobacterium tumefaciens strain C58

Hybridoma cell lines were derived from the fusion of mouse myeloma cells with spleen cells from a mouse that had been immunized withAgrobacterium tumefaciens strain A759, a flagella-less mutant of strain C58 containing the Ti plasmid of strain B6. All of the 20 antibodies produced by the cloned hybridomas reacted with strain C58 and with other strains derived from C58. The antibodies did not react with 34 other strains ofA. tumefaciens, representing the three biovars, or with strains ofA. radiobacter, A. rubi, Rhizobium leguminosarum, R. meliloti, or other plant-associated bacteria such asErwinia herbicola andPseudomonas syringae. In addition to reacting with whole cells of strain A759, the antibodies reacted with phenol-water extracts of A759, indicating that they may recognize the lipopolysaccharide. These antibodies may be useful for ecological and epidemiological studies ofA. tumefaciens strain C58 in the agroecosystem.     

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.     

SyrD is required for syringomycin production by Pseudomonas syringae pathovar syringae and is related to a family of ATP-binding secretion proteins

The syrD gene of Pseudomonas syringae pathovar syringae strain B301D-R was characterized and sequenced. The syrD open reading frame is 1695bp long and encodes a predicted protein, SyrD, of =63kDa. Database searches revealed that SyrD shares a high degree of similarity with the ATP-binding cassette (ABC) superfamily of transporter proteins which are responsible for specific nutrient uptake and for secretion of certain cellular products in prokaryotes, and for multiple drug resistance in mammals. The amino acid sequence homology between SyrD and the ABC proteins was greatest at the conserved residues which constitute the ATP-binding cassette of these proteins; these residues lie in the hydrophilic C-terminal half of SyrD. The N-terminus of SyrD is predicted to be hydrophobic and to contain six membrane-spanning α-helices. syrD mutants of strain B301D-R were significantly less virulent than other syr mutants, were deficient in four large polypeptides thought to be components of a syringomycin synthetase complex, and showed reduced expression of a syrB-lacZ reporter gene fusion in trans. It is proposed that SyrD is a cyto-plasmic membrane protein that functions as an ATP-driven efflux pump for the secretion of syringomycin.     

Plasmids of serogroup 1 strains ofLegionella pneumophila

Twelve strains ofLegionella pneumophila were tested for the presence of plasmid DNA. Three strains, belonging to serogroup 1, had large plasmids of 83.8×10⁶ daltons, as determined by electron microscopy. A fourth strain, also from serogroup 1, had a similar large plasmid in addition to a smaller plasmid. Restriction analysis of plasmid DNA isolated from the strains with a single size plasmid indicated that the plasmids were structurally very similar. The biologic functions of these plasmids are yet to be determined.     

Plasmids in clinical isolates ofStreptococcus pneumoniae

Forty clinical isolates ofStreptococcus pneumoniae with various antibiotic resistance profiles were screened for the presence of plasmids. Plasmids were demonstrated in five isolates. Three procedures for plasmid isolation were evaluated. A 10.8-kb plasmid was demonstrated by all three methods, but a further four plasmids were detected with one method only. The sizes of these plasmids were 11.5 kb, 10.8 kb, and 3.0 kb (two strains). Curing experiments were performed, but no plasmid/antibiotic correlation was observed. Tetracycline, erythromycin, and clindamycin resistance was lost in one strain, although the plasmid was still present.     

Heterologous expression of the gene for the ethylene-forming enzyme fromPseudomonas syringae in the cyanobacteriumSynechococcus

Summary Bioconversion of atmospheric carbon dioxide to ethylene was studied in a recombinant cyanobacterium. The gene for the ethylene-forming enzyme ofPseudomonas syringae pv.phaseolicola PK2 was cloned and expressed in the cyanobacteriumSynechococcus PCC7942 R2-SPc by use of a shuttle vector pUC303. The ethylene-forming activityin vivo ofSynechococcus PCC7942 R2-SPc that carried the gene for the ethylene-forming enzyme ofP. syringae pv.phaseolicola PK2 was one-fifth of that ofE. coli JM109 that harbored the same plasmid. The enzyme accounted for 0.021% by weight of the total soluble protein inSynechococcus PCC7942 R2-SPc.     

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     

Methods of assessment of transfer of a gentamycin-resistance gene (aacC1) from genetically engineered microorganisms into agriculturally important soil bacteria

In order to assess the risk associated with the deliberate release of genetically engineered microorganisms (GEMs) into the agricultural environment, the transfer of plasmids between bacterial strains was investigated under laboratory conditions. Genetically modified Rhizobium leguminosarum and Agrobacterium tumefaciens strains carrying the gentamycin acetyltransferase resistance gene (aacC1) on various plasmids were investigated for their ability to transfer the aacC1 gene to their wild-type (w.t.) counterparts, as well as to Pseudomonas syringae. Conjugation experiments between the various strains, were carried out after the relevant characteristics and conditions for selective growth of each bacterial strain had been ascertained. After conjugations on filters had been completed, the putative transconjugants were grown in media containing antibiotics and assessed for the presence of aacC1 gene by: (a) DNA plasmid profile; (b) expression of AAC(3)-I enzyme activity; (c) colony hybridization using a ³²P-labelled DNA probe complementary to the aacC1 gene. The results obtained indicate that transfer of the aacC1 gene from genetically modified strains of R. leguminosarum into a plasmid-free strain of A. tumefaciens occurred via self-transmissible plasmids. Alternatively, genetically modified A. tumefaciens bearing the aacC1 gene on plasmids acquired from R. leguminosarum strains, transferred it ineffectively to a hardly detectable frequency. No transfer of the aacC1 gene from genetically modified R. leguminosarum or A. tumefaciens strains into P. syringae has been observed. These data indicate that in the absence of the RP4 element, genetically modified A. tumefaciens is not able to efficiently transfer aacC1 into w.t. R. leguminosarum and P. syringae. Correspondence to: A. S. Tsiftsoglou