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Papers by Alain Bultreys

Conventional identification tests and rapid tests based on phytotoxin and pyoverdin production we... more Conventional identification tests and rapid tests based on phytotoxin and pyoverdin production were used to determine the diversity in 170 fluorescent and non-fluorescent oxidase-negative strains isolated from diseased plant material in pear, sweet cherry, sour cherry and plum orchards in Belgium. Determination of phytotoxin production was based on a biological test for the detection of toxic lipodepsipeptide production and PCR tests to detect the syrB, syrD and cfl genes. The pyoverdin tests were visual, spectrophotometrical and isoelectric focalisation tests; they detected the Fe(III)-chelated atypical pyoverdin produced by Pseudomonas syringae. The P. s. pv. syringae and P. s. pv. morsprunorum were almost exclusively detected in pear and sweet cherry orchards and the phytotoxin tests proved useful in identifying these pathovars, although caution was needed regarding negative results because a few strains were unable to produce phytotoxins. In contrast, many plum and sour cherry strains could not be affiliated to a pathovar. All the non-toxin-producing strains investigated in this study were fluorescent. The pyoverdin tests classified them in the siderovar of P. syringae. The results therefore underscored the great variation in the P. syringae strains isolated from plum and sour cherry orchards. The pyoverdin tests were particularly useful in identifying the non-toxin-producers and they showed a real potential as general identification tools of fluorescent P. syringae strains, but they were less discriminatory than the phytotoxin tests. Also, the pyoverdin tests could not identify non-fluorescent strains comparable to 34 of the 45 P. s. pv. morsprunorum strains in this study.

Applied and Environmental Microbiology, Feb 1, 2003

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopatho... more The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two ␤-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.

Plant Pathology, 2018

Fosetyl-Al (the aluminium salt of ethyl-phosphite) is an acid product used to fight oomycete dise... more Fosetyl-Al (the aluminium salt of ethyl-phosphite) is an acid product used to fight oomycete diseases sometimes used against bacterial diseases, but its antibacterial mode of action is largely unknown. Consequently, the direct effects of fosetyl-Al and neutralized fosetyl-Al on the colonization of leaf surfaces by Pseudomonas syringae were compared. Control of colonization was highest and almost complete in acidic conditions (99.7%), showing the importance of acidity for maximal antibacterial efficacy on plant surfaces. However, the high inhibition obtained with neutralized fosetyl-Al (88.9%) suggested another direct effect. Therefore, it was investigated in vitro whether ethyl-phosphite has antibacterial activity, possibly related to its phosphite content. Inhibition of P. syringae growth by ethyl-phosphite was observed and influenced by the carbon source and by phosphate. Growth inhibition reached 83.1% in unshaken conditions in a phosphatedepleted glucose medium. Phosphite drastically increased the effects and induced up to 99.7% growth inhibition. Mass spectrometric analysis indicated that P. syringae hydrolyzed ethyl-phosphite to phosphite using a cell-linked phosphatase activity. Specific phosphite toxicity was observed in phosphate-sufficient succinate media and phosphite-induced phosphorus starvation was observed in phosphate-depleted glucose media, indicating that ethyl-phosphite and phosphite have direct environmental impacts on bacteria. Phosphite toxicity for P. syringae probably occurred in the phyllosphere. The study indicates the potential and limitations of fosetyl-Al as a direct antibacterial product and helps in understanding the fosetyl-Al control of flower bud blast in pear. It raises the prospect of using phosphite in plant bacterial disease control.

New Disease Reports, 2020

Phytoma-La Défense des végétaux, 2012

Zeitschrift für Naturforschung C, 2004

The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide sid... more The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide side chain originally produced by the closely related species Pseudomonas syringae and P. cichorii is reported. Mass spectrometry and nuclear magnetic resonance analyses as well as the determination of the configuration of the amino acids after degradation indicate that these two pyoverdins differ only by the replacement of the first in-chain serine by glycine. The pyoverdins of P. syringae and P. cichorii and the dihydropyoverdin of P. syringae can be used by both species as siderophores.

L'étude de la production de phytotoxines et de sidérophores de type pyoverdine par des souche... more L'étude de la production de phytotoxines et de sidérophores de type pyoverdine par des souches de Pseudomonassyringae pv. syringae et de P. syringae pv. morsprunorum a été réalisée en vue de caractériser les souches concernant la capacité de production de ces types de métabolites secondaires et en vue de déterminer dans quelle mesure ces métabolites permettent de différencier les pathovars, de les classifier, voire de les identifier.Les conditions favorisant la production de lipodepsipeptides toxiques et de pyoverdines ont été étudiées. Des milieux de culture et des techniques de production ont été définis en culture solide et en culture liquide. Les résultats de ces travaux permettent de penser que les conditions nécessaires à la production de ces métabolites par P. syringae sont probablement régulièrement rencontrées sur la plante.Un test biologique détectant la capacité de production de lipodepsipeptides toxiques et un test PCR détectant le gène syrDimpliqué dans leur product...

Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics, 2008

ABSTRACT Pseudomonas syringae was isolated from lesions found in cortical tissues of Belgian hors... more ABSTRACT Pseudomonas syringae was isolated from lesions found in cortical tissues of Belgian horse-chestnut (Aesculus hippocastanum) trunks. A collection of about 50 strains was established from 6 sites in Brussels and 11 sites in Wallonia, but the pathogen was not found in the South-East of Belgium. The strains were identified by pyoverdin tests, induction of potato rot, the cfl PCR test, and REPPCR. The investigated strains were highly virulent on horse-chestnut detached twigs collected in winter and summer, although the propagation outside the inoculated zones was reduced in summer. The accentuated propagation during winter was confirmed by inoculation of 20 young horse-chestnut trunks: in the end of winter and early spring, a mean progression in the cortical tissues of 3.45 mm in 48 days was observed (0.05 mm for the controls), whereas the progression during spring and summer was reduced by a factor seven. In this latter period, canker formations were observed; also, P. syringae was still isolable from naturally infected older trunks. Comparison of REP-PCR profiles were carried out with 60 pathovars of P. syringae and with Pseudomonas viridiflava,Pseudomonas meliae, Pseudomonas ficuserectae,Pseudomonas cannabina and Pseudomonas tremae. REP- and ERICPCR analyses indicated the relatedness of the horse-chestnut strains, although site-related genetic groups were observed, as well as genetic similarities and differences with P. syringae pv. morsprunorum race 1, pv. aesculi, pv. cunninghamiae and pv. daphniphylli; both the pathovars morsprunorum race 1 and aesculi possessed the cfl gene and all the investigated horse-chestnut strains produced coronatine. Only strains from horse-chestnut and pathovar aesculi were similarly virulent on A. hippocastanum. The data indicate that P. syringae strains are living on A. hippocastanum for a long time and would agree with their grouping with P. syringae pv. aesculi inducing leaf spots on Aesculus indica, but this pathovar would then be genetically heterogeneous.

Soil Biology, 2007

... Pseudomonine production was detected by RT-PCR (Mercado-Blanco et al. ... On the other hand, ... more ... Pseudomonine production was detected by RT-PCR (Mercado-Blanco et al. ... On the other hand, the pyoverdin of P. syringae is not involved in virulence in cherry fruits (Cody and Gross 1987), but its production is stimulated in conditions found on plant sur-face when P. syringae ...

Applied and environmental microbiology, 2003

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopatho... more The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produ...

Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics, 2008

ABSTRACT The diversifying evolution observed in pyoverdin genes and the presence of numerous side... more ABSTRACT The diversifying evolution observed in pyoverdin genes and the presence of numerous siderophore membrane receptors indicates that iron-supplying systems are important for fitness of the fluorescent pseudomonads. It is useful for a bacterium to produce a siderophore unusable by others and siderophores, or siderophore genes, that are specific to certain bacteria have been selected during evolution; they can therefore be used in identification. Pyoverdins are the principal siderophores of fluorescent pseudomonads and they contain a variable peptide chain. Because of the evolution in this peptide chain, the pyoverdins of Pseudomonas cichorii and P. syringae, and related species, are differentiable by visual and spectrophotometrical analysis from pyoverdins produced by species belonging to the saprophytic fluorescent Pseudomonas group. The common pyoverdin of P. syringae and related species P. viridiflava and P. ficuserectae has been shown to be specific. This characteristic can therefore be used for presumptive identification of these species by HPLC analysis of their pyoverdin. This significantly reduces the number of identification tests required. Additionally, these approaches worked well to rapidly discriminate among fluorescent strains in early stages of isolation, and to identify strains of P. viridiflava and of P. syringae pathogenic on fruit and horse-chestnut trees. P. syringae pathovars antirrhini,apii, avii, berberidis,delphinii, lachrymans,passiflorae, persicae,tomato,maculicola,viburni,helianthi,tagetis,theae and morsprunorum race 2 (genospecies 3, 7 or 8), as well as P. phaseolicola and P. glycinea (genospecies 2) possess an irp1 gene involved in the production of the yersiniabactin siderophore. However, as yersiniabactin production is not systematically observed by HPLC, an irp1-based PCR test was used for these pathovars.

Applied and environmental microbiology, 1999

Toxin-based identification procedures are useful for differentiating Pseudomonas syringae pathova... more 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 t...

Journal of Plant Pathology, 2010

Bacterial canker of stone fruits caused by Pseudomonas syringae pv. syringae and pv. morsprunorum... more Bacterial canker of stone fruits caused by Pseudomonas syringae pv. syringae and pv. morsprunorum has been studied for a century now and this research brought decisive advances in the understanding of the epidemiology of foliar bacterial diseases and epiphytic stages in the life cycles of plant pathogenic bacteria. The economic importance of the disease and lack of effective control measures stimulated recent studies on the ecology of the pathogens and their characterization and identification. New techniques of identification are based on the ecologically and pathologically important siderophores pyoverdine and yersiniabactine, and phytotoxins including coronatine and toxic lipodepsipeptides. Genetic methods based on total DNA homology, repeated sequences (rep-PCR), melting profiles (PCR MP) and DNA sequencing (MLST) proved the existence of three distinct pathogens: races 1 and 2 of pathovar morsprunorum, each genetically homogenous, and pathovar syringae, highly heterogeneous. In both pathovars, genetic groups differing in their pathogenicity were determined and isolate-host relationships pointed out.

Applied and Environmental Microbiology, 2000

The production of peptide siderophores and the variation in siderophore production among strains ... more 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.

Applied and Environmental Microbiology, 2000

The production of peptide siderophores and the variation in siderophore production among strains ... more 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

European Journal of Plant Pathology, 2010

The pathogenicity of 99 Belgian Pseudomonas syringae strains representative of the genetic divers... more The pathogenicity of 99 Belgian Pseudomonas syringae strains representative of the genetic diversity encountered in Belgian fruit orchards was evaluated by using 17 pathogenicity tests conducted on pear, cherry, plum, lilac, sugar beet and wheat. The P. syringae pv. morsprunorum strains were pathogenic to stone fruit species but the race 1 strains possessing the cfl gene involved in coronatine production were pathogenic in more tests than those lacking the gene. Also, sweet cherry twigs were a better material to detect pathogenic strains of race 1 and sour cherry twigs of race 2, which accorded with race 2 presence in sour cherry orchards in Belgium. Three groups were defined in the pv. syringae based on pathogenicity. One group pathogenic in 71.1% of the tests and to lilac included toxic lipodesipeptide-producing (TLP+) strains. The second group pathogenic in 26.8% of the tests and non-pathogenic to lilac included TLP+ strains. The thirth group pathogenic in 9.1% of the tests and almost specifically pathogenic to pear included TLP− strains. The three groups were genetically heterogeneous. Although strain-host relationships were noted within the pv. syringae, aptata and atrofaciens when considering the strain origins, such relationships were not found in the pathogenicity tests, suggesting that pathogenicity tests could probably not reproduce all the aspects of the host-pathogen interactions. None of the pathogenicity tests was able to provide all the information provided by the complete study. A test on pear buds indicated that strains different from the pv. syringae were pathogenic to pear.

Zeitschrift für Naturforschung C, 2004

The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide sid... more The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide side chain originally produced by the closely related species Pseudomonas syringae and P. cichorii is reported. Mass spectrometry and nuclear magnetic resonance analyses as well as the determination of the configuration of the amino acids after degradation indicate that these two pyoverdins differ only by the replacement of the first in-chain serine by glycine. The pyoverdins of P. syringae and P. cichorii and the dihydropyoverdin of P. syringae can be used by both species as siderophores.

Conventional identification tests and rapid tests based on phytotoxin and pyoverdin production we... more Conventional identification tests and rapid tests based on phytotoxin and pyoverdin production were used to determine the diversity in 170 fluorescent and non-fluorescent oxidase-negative strains isolated from diseased plant material in pear, sweet cherry, sour cherry and plum orchards in Belgium. Determination of phytotoxin production was based on a biological test for the detection of toxic lipodepsipeptide production and PCR tests to detect the syrB, syrD and cfl genes. The pyoverdin tests were visual, spectrophotometrical and isoelectric focalisation tests; they detected the Fe(III)-chelated atypical pyoverdin produced by Pseudomonas syringae. The P. s. pv. syringae and P. s. pv. morsprunorum were almost exclusively detected in pear and sweet cherry orchards and the phytotoxin tests proved useful in identifying these pathovars, although caution was needed regarding negative results because a few strains were unable to produce phytotoxins. In contrast, many plum and sour cherry strains could not be affiliated to a pathovar. All the non-toxin-producing strains investigated in this study were fluorescent. The pyoverdin tests classified them in the siderovar of P. syringae. The results therefore underscored the great variation in the P. syringae strains isolated from plum and sour cherry orchards. The pyoverdin tests were particularly useful in identifying the non-toxin-producers and they showed a real potential as general identification tools of fluorescent P. syringae strains, but they were less discriminatory than the phytotoxin tests. Also, the pyoverdin tests could not identify non-fluorescent strains comparable to 34 of the 45 P. s. pv. morsprunorum strains in this study.

Applied and Environmental Microbiology, Feb 1, 2003

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopatho... more The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two ␤-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.

Plant Pathology, 2018

Fosetyl-Al (the aluminium salt of ethyl-phosphite) is an acid product used to fight oomycete dise... more Fosetyl-Al (the aluminium salt of ethyl-phosphite) is an acid product used to fight oomycete diseases sometimes used against bacterial diseases, but its antibacterial mode of action is largely unknown. Consequently, the direct effects of fosetyl-Al and neutralized fosetyl-Al on the colonization of leaf surfaces by Pseudomonas syringae were compared. Control of colonization was highest and almost complete in acidic conditions (99.7%), showing the importance of acidity for maximal antibacterial efficacy on plant surfaces. However, the high inhibition obtained with neutralized fosetyl-Al (88.9%) suggested another direct effect. Therefore, it was investigated in vitro whether ethyl-phosphite has antibacterial activity, possibly related to its phosphite content. Inhibition of P. syringae growth by ethyl-phosphite was observed and influenced by the carbon source and by phosphate. Growth inhibition reached 83.1% in unshaken conditions in a phosphatedepleted glucose medium. Phosphite drastically increased the effects and induced up to 99.7% growth inhibition. Mass spectrometric analysis indicated that P. syringae hydrolyzed ethyl-phosphite to phosphite using a cell-linked phosphatase activity. Specific phosphite toxicity was observed in phosphate-sufficient succinate media and phosphite-induced phosphorus starvation was observed in phosphate-depleted glucose media, indicating that ethyl-phosphite and phosphite have direct environmental impacts on bacteria. Phosphite toxicity for P. syringae probably occurred in the phyllosphere. The study indicates the potential and limitations of fosetyl-Al as a direct antibacterial product and helps in understanding the fosetyl-Al control of flower bud blast in pear. It raises the prospect of using phosphite in plant bacterial disease control.

New Disease Reports, 2020

Phytoma-La Défense des végétaux, 2012

Zeitschrift für Naturforschung C, 2004

The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide sid... more The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide side chain originally produced by the closely related species Pseudomonas syringae and P. cichorii is reported. Mass spectrometry and nuclear magnetic resonance analyses as well as the determination of the configuration of the amino acids after degradation indicate that these two pyoverdins differ only by the replacement of the first in-chain serine by glycine. The pyoverdins of P. syringae and P. cichorii and the dihydropyoverdin of P. syringae can be used by both species as siderophores.

L'étude de la production de phytotoxines et de sidérophores de type pyoverdine par des souche... more L'étude de la production de phytotoxines et de sidérophores de type pyoverdine par des souches de Pseudomonassyringae pv. syringae et de P. syringae pv. morsprunorum a été réalisée en vue de caractériser les souches concernant la capacité de production de ces types de métabolites secondaires et en vue de déterminer dans quelle mesure ces métabolites permettent de différencier les pathovars, de les classifier, voire de les identifier.Les conditions favorisant la production de lipodepsipeptides toxiques et de pyoverdines ont été étudiées. Des milieux de culture et des techniques de production ont été définis en culture solide et en culture liquide. Les résultats de ces travaux permettent de penser que les conditions nécessaires à la production de ces métabolites par P. syringae sont probablement régulièrement rencontrées sur la plante.Un test biologique détectant la capacité de production de lipodepsipeptides toxiques et un test PCR détectant le gène syrDimpliqué dans leur product...

Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics, 2008

ABSTRACT Pseudomonas syringae was isolated from lesions found in cortical tissues of Belgian hors... more ABSTRACT Pseudomonas syringae was isolated from lesions found in cortical tissues of Belgian horse-chestnut (Aesculus hippocastanum) trunks. A collection of about 50 strains was established from 6 sites in Brussels and 11 sites in Wallonia, but the pathogen was not found in the South-East of Belgium. The strains were identified by pyoverdin tests, induction of potato rot, the cfl PCR test, and REPPCR. The investigated strains were highly virulent on horse-chestnut detached twigs collected in winter and summer, although the propagation outside the inoculated zones was reduced in summer. The accentuated propagation during winter was confirmed by inoculation of 20 young horse-chestnut trunks: in the end of winter and early spring, a mean progression in the cortical tissues of 3.45 mm in 48 days was observed (0.05 mm for the controls), whereas the progression during spring and summer was reduced by a factor seven. In this latter period, canker formations were observed; also, P. syringae was still isolable from naturally infected older trunks. Comparison of REP-PCR profiles were carried out with 60 pathovars of P. syringae and with Pseudomonas viridiflava,Pseudomonas meliae, Pseudomonas ficuserectae,Pseudomonas cannabina and Pseudomonas tremae. REP- and ERICPCR analyses indicated the relatedness of the horse-chestnut strains, although site-related genetic groups were observed, as well as genetic similarities and differences with P. syringae pv. morsprunorum race 1, pv. aesculi, pv. cunninghamiae and pv. daphniphylli; both the pathovars morsprunorum race 1 and aesculi possessed the cfl gene and all the investigated horse-chestnut strains produced coronatine. Only strains from horse-chestnut and pathovar aesculi were similarly virulent on A. hippocastanum. The data indicate that P. syringae strains are living on A. hippocastanum for a long time and would agree with their grouping with P. syringae pv. aesculi inducing leaf spots on Aesculus indica, but this pathovar would then be genetically heterogeneous.

Soil Biology, 2007

... Pseudomonine production was detected by RT-PCR (Mercado-Blanco et al. ... On the other hand, ... more ... Pseudomonine production was detected by RT-PCR (Mercado-Blanco et al. ... On the other hand, the pyoverdin of P. syringae is not involved in virulence in cherry fruits (Cody and Gross 1987), but its production is stimulated in conditions found on plant sur-face when P. syringae ...

Applied and environmental microbiology, 2003

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopatho... more The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produ...

Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics, 2008

ABSTRACT The diversifying evolution observed in pyoverdin genes and the presence of numerous side... more ABSTRACT The diversifying evolution observed in pyoverdin genes and the presence of numerous siderophore membrane receptors indicates that iron-supplying systems are important for fitness of the fluorescent pseudomonads. It is useful for a bacterium to produce a siderophore unusable by others and siderophores, or siderophore genes, that are specific to certain bacteria have been selected during evolution; they can therefore be used in identification. Pyoverdins are the principal siderophores of fluorescent pseudomonads and they contain a variable peptide chain. Because of the evolution in this peptide chain, the pyoverdins of Pseudomonas cichorii and P. syringae, and related species, are differentiable by visual and spectrophotometrical analysis from pyoverdins produced by species belonging to the saprophytic fluorescent Pseudomonas group. The common pyoverdin of P. syringae and related species P. viridiflava and P. ficuserectae has been shown to be specific. This characteristic can therefore be used for presumptive identification of these species by HPLC analysis of their pyoverdin. This significantly reduces the number of identification tests required. Additionally, these approaches worked well to rapidly discriminate among fluorescent strains in early stages of isolation, and to identify strains of P. viridiflava and of P. syringae pathogenic on fruit and horse-chestnut trees. P. syringae pathovars antirrhini,apii, avii, berberidis,delphinii, lachrymans,passiflorae, persicae,tomato,maculicola,viburni,helianthi,tagetis,theae and morsprunorum race 2 (genospecies 3, 7 or 8), as well as P. phaseolicola and P. glycinea (genospecies 2) possess an irp1 gene involved in the production of the yersiniabactin siderophore. However, as yersiniabactin production is not systematically observed by HPLC, an irp1-based PCR test was used for these pathovars.

Applied and environmental microbiology, 1999

Toxin-based identification procedures are useful for differentiating Pseudomonas syringae pathova... more 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 t...

Journal of Plant Pathology, 2010

Bacterial canker of stone fruits caused by Pseudomonas syringae pv. syringae and pv. morsprunorum... more Bacterial canker of stone fruits caused by Pseudomonas syringae pv. syringae and pv. morsprunorum has been studied for a century now and this research brought decisive advances in the understanding of the epidemiology of foliar bacterial diseases and epiphytic stages in the life cycles of plant pathogenic bacteria. The economic importance of the disease and lack of effective control measures stimulated recent studies on the ecology of the pathogens and their characterization and identification. New techniques of identification are based on the ecologically and pathologically important siderophores pyoverdine and yersiniabactine, and phytotoxins including coronatine and toxic lipodepsipeptides. Genetic methods based on total DNA homology, repeated sequences (rep-PCR), melting profiles (PCR MP) and DNA sequencing (MLST) proved the existence of three distinct pathogens: races 1 and 2 of pathovar morsprunorum, each genetically homogenous, and pathovar syringae, highly heterogeneous. In both pathovars, genetic groups differing in their pathogenicity were determined and isolate-host relationships pointed out.

Applied and Environmental Microbiology, 2000

The production of peptide siderophores and the variation in siderophore production among strains ... more 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.

Applied and Environmental Microbiology, 2000

The production of peptide siderophores and the variation in siderophore production among strains ... more 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

European Journal of Plant Pathology, 2010

The pathogenicity of 99 Belgian Pseudomonas syringae strains representative of the genetic divers... more The pathogenicity of 99 Belgian Pseudomonas syringae strains representative of the genetic diversity encountered in Belgian fruit orchards was evaluated by using 17 pathogenicity tests conducted on pear, cherry, plum, lilac, sugar beet and wheat. The P. syringae pv. morsprunorum strains were pathogenic to stone fruit species but the race 1 strains possessing the cfl gene involved in coronatine production were pathogenic in more tests than those lacking the gene. Also, sweet cherry twigs were a better material to detect pathogenic strains of race 1 and sour cherry twigs of race 2, which accorded with race 2 presence in sour cherry orchards in Belgium. Three groups were defined in the pv. syringae based on pathogenicity. One group pathogenic in 71.1% of the tests and to lilac included toxic lipodesipeptide-producing (TLP+) strains. The second group pathogenic in 26.8% of the tests and non-pathogenic to lilac included TLP+ strains. The thirth group pathogenic in 9.1% of the tests and almost specifically pathogenic to pear included TLP− strains. The three groups were genetically heterogeneous. Although strain-host relationships were noted within the pv. syringae, aptata and atrofaciens when considering the strain origins, such relationships were not found in the pathogenicity tests, suggesting that pathogenicity tests could probably not reproduce all the aspects of the host-pathogen interactions. None of the pathogenicity tests was able to provide all the information provided by the complete study. A test on pear buds indicated that strains different from the pv. syringae were pathogenic to pear.

Zeitschrift für Naturforschung C, 2004

The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide sid... more The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide side chain originally produced by the closely related species Pseudomonas syringae and P. cichorii is reported. Mass spectrometry and nuclear magnetic resonance analyses as well as the determination of the configuration of the amino acids after degradation indicate that these two pyoverdins differ only by the replacement of the first in-chain serine by glycine. The pyoverdins of P. syringae and P. cichorii and the dihydropyoverdin of P. syringae can be used by both species as siderophores.