Louis Gardan

Auxin Production Is a Common Feature of Most Pathovars of Pseudomonas syringae

We investigated indole-3-acetic acid (IAA) production by 57 pathovars of Pseudomonas syringae and related species. Most of those analyzed produced IAA, especially in the presence of tryptophan. Eight strains produced high IAA concentrations in the absence of Trp. The iaaM and iaaH genes of P. savastanoi pv. savastanoi were detected in a limited number of strains only, including the eight above-mentioned strains. Thus, IAA synthesis in most assayed strains of P. syringae and related species does not involve genes highly similar to iaaM and iaaH. In contrast, the iaaL gene encoding an IAA-lysine synthase was detected in most pathovars, and was often found on plasmids.

Siderophore Typing, a Powerful Tool for the Identification of Fluorescent and Nonfluorescent Pseudomonads

ABSTRACT A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, “Pseudomonas mosselii,” “Pseudomonas palleronii,” Pseudomonas rhodesiae, “Pseudomonas salomonii,” Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.

DNA relatedness among strains of Streptomyces pathogenic to potato in France: description of three new species, S. europaeiscabiei sp. nov. and S. stelliscabiei sp. nov. associated with common scab, and S. reticuliscabiei sp. nov. associated with netted scab.

The genomic relatedness was evaluated by DNA-DNA hybridization for 23 strains (21 were pathogenic and two were saprophytic strains) isolated from lesions of common and netted scab in France and 19 strains from other countries, including type strains of Streptomyces species. Three genomospecies were defined within the conventional species of Streptomyces scabies, and these genomospecies were different from other pathogenic described species (Streptomyces acidiscabies, Streptomyces caviscabies) based on previously published phenotypic data. Two of these genomospecies (1 and 3) correspond to new species, for which the names Streptomyces europaeiscabiei sp. nov. (with type strain CFBP 4497T) and Streptomyces stelliscabiei sp. nov. (with type strain CFBP 4521T) are proposed. Genomospecies 2 corresponds to S. scabies (with type strain CFBP 4517T = ATCC 49173T), and includes only one French strain. The pathogenic strains associated with netted scab lesions constituted a new species that was named Streptomyces reticuliscabiei sp. nov. (with type strain CFBP 4531T). The G+C content of DNA from the three strains CFBP 4497T (S. europaeiscabiei), CFBP 4521T (S. stelliscabiei), CFBP 4531T (S. reticuliscabiei) was 71.3, 71.0 and 69.8 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that the type strain CFBP 4497T was very similar to the type strain of S. scabies, whereas, the type strain of S. stelliscabiei, CFBP 4521T, was very similar to the type strain of Streptomyces bottropensis. On the basis of 16S rRNA gene sequences, the type strain of S. reticuliscabiei, CFBP 4531T, differed extensively from the other strains of Streptomyces tested.

Pseudomonas brassicacearum sp. nov. and Pseudomonas thivervalensis sp. nov., two root-associated bacteria isolated from Brassica napus and Arabidopsis thaliana

Bacteria isolates phenotypically related to Pseudomonas corrugata have frequently been isolated from the rhizosphere of Arabidopsis thaliana and Brassica napus grown on different soils. 16S rDNA (rrs) gene sequencing, DNA-DNA hybridization, biochemical characterization and siderophore typing showed that these isolates belong to two different species that are distinct from other species of the genus Pseudomonas, including P. corrugata. A description of properties of these two new species is given based on the study of 16 isolates. Proposed names are Pseudomonas brassicacearum (10 strains studied) and Pseudomonas thivervalensis (6 strains studied). The type strain of Pseudomonas brassicacearum is CFBP 11706T and that of Pseudomonas thivervalensis is CFBP 11261T.

Phenotypic and genomic evidence for the revision of Pseudomonas corrugata and proposal of Pseudomonas mediterranea sp. nov.

To re-examine the taxonomic status of Pseudomonas corrugata, 27 strains of this species were studied using a polyphasic approach. Numerical analysis of phenotypic data revealed two phena, A (including the P. corrugata type strain) and B, which could be clearly differentiated by the assimilation of mesotartrate, 2-ketogluconate and histamine. The mean DNA reassociation values with labelled DNA of P. corrugata type strain CFBP 2431T (phenon A) and strain CFBP 5447T (phenon B) were high for strains belonging to the same phenon (96.9 and 98.5%, respectively), whereas the DNA relatedness between the two phena was assessed as being close to 70%, which represents the value that is accepted for the definition of a bacterial species. Phena A and B were also differentiated by means of DNA profiles generated by heteroduplex mobility assay of PCR products of 16S rDNA hypervariable region 2, HaeIII restriction of the amplified internal transcribed spacer, REP- and BOX-PCR profiles, and by PCR with two pairs of specific primers. A comparison of the 16S rRNA sequences of strains CFBP 5447T and CFBP 5458 from phenon B with the available sequences of Pseudomonas species showed that these strains formed a cluster distinct from the P. corrugata type strain. Thus, a new species, Pseudomonas mediterranea, is proposed for strains of phenon B. The type strain is strain CFBP 5447T (= ICMP 14184T); its G+C content is 60.2 mol%.

Taxonomy of Pseudomonas strains isolated from tomato pith necrosis: emended description of Pseudomonas corrugata and proposal of three unnamed fluorescent Pseudomonas genomospecies.

Thirty-three fluorescent Pseudomonas strains isolated from tomato pith necrosis (FPTPN strains) and 89 Pseudomonas corrugata strains were studied by numerical taxonomy. In the dendrogram of distances, the P. corrugata strains constituted a single phenon (phenon 1), whereas 17 of the 33 FPTPN strains clustered in a separate phenon (phenon 2). The other 16 FPTPN strains were included in phena consisting of well-characterized fluorescent Pseudomonas species or were isolated phenotypes. Phena 1 and 2 were distinguished by fluorescence on King B medium, accumulation of poly-beta-hydroxybutyrate, production of levan, and assimilation of sorbitol. DNA-DNA hybridization showed that P. corrugata is a true genomic species (66 to 100% DNA relatedness) and that the FPTPN strains of phenon 2 were divided into three genomic groups. Genomic groups 1 and 2 were not distinct from each other phenotypically, and genomic group 3 could be distinguished from genomic groups 1 and 2 only on the basis of assimilation of citraconate and laevulinate. Genomic groups 1 and 2 are related to P. corrugata (40 to 55% DNA relatedness), whereas genomic group 3 is less closely related to P. corrugata (20 to 23% DNA relatedness). The lipopolysaccharide patterns on electrophoresis gels and fatty acid profiles of strains belonging to genomic group 1 through 3 are different from each other and from the lipopolysaccharide patterns and fatty acid profiles of P. corrugata. However, cross-reactions were observed between P. corrugata and the FPTPN strain genomic groups, indicating that there are common epitopes of the lipopolysaccharides. The three FPTPN strain genomic groups were not named as species but were designated Pseudomonas genomospecies FP1, FP2, and FP3.

The Relationship of Host Range, Physiology, and Genotype to Virulence on Cantaloupe in Pseudomonas syringae from Cantaloupe Blight Epidemics in France.

ABSTRACT In 1993, a bacterial blight caused important losses of cantaloupe (Cucumis melo var. cantalupensis) in southwestern France and has now been reported in all cantaloupe-growing regions of France. The causal agent of this blight is Pseudomonas syringae, although on a worldwide basis this bacterium has not been a major pathogen of melon for over 50 years. To identify the pathovar of the cantaloupe pathogen, we employed biochemical tests, plasmid and chromosomal profiling, and host range studies for 23 strains from cantaloupe and 47 reference strains of 14 pathovars of P. syringae. Numerical analysis of 119 traits, serological typing, syringomycin production, and BOX-polymerase chain reaction profiles did not allow us to differentiate among pathovars related to P. syringae pv. syringae. Host range studies of cantaloupe and references strains on 18 plant species showed that virulence to sugar beet was a common feature of strains virulent on cantaloupe, but was not common to strains avirulent on cantaloupe. Virulence to other species of plants varied among strains, but the overall extent of the host range was proportional to aggressiveness to cantaloupe. We propose that the strains attacking cantaloupe in France be considered P. syringae pv. aptata and that adequate host range testing may reveal that this pathovar is the cause of cantaloupe blight reported in other parts of the world.

Multiphasic approach for the identification of the different classification levels of Pseudomonas savastanoi pv. phaseolicola

The relationships between strains of Pseudomonas savastanoi pv. phaseolicola (P. sav. phaseolicola), P. syringae pv. tabaci (P. syr. tabaci) and P. syr. syringae which all cause disease on bean; the related species P. sav. glycinea and P. syr. actinidiae, and reference bacteria, were evaluated by studying the phenotypic and genetic diversity of a collection of 62 strains. All the P. sav. phaseolicola strains tested produced characteristic watersoaked lesions on bean pods. Other pathovars produced varying combinations of symptoms including necrotic lesions, with or without watersoaked centres and sunken tissue collapse of the lesion (P. syr. tabaci) and necrotic lesions with or without sunken collapse (P. syr. syringae). At the genomospecies level, all the strains of P. sav. phaseolicola, P. sav. glycinea and P. syr. tabaci, belonging to genomospecies 2, could be separated from P. syr. syringae strains (genomospecies 1) and P. syr. actinidiae strains (unknown genomospecies) by BOX-PCR and DNA/DNA hybridisation. To distinguish P. sav. phaseolicola, within genomospecies 2, from P. sav. glycinea and P. syr. tabaci, it was necessary to perform nutritional characterisations myo-inositol negative and p-hydroxy benzoate positive for P. sav. phaseolicola strains), PCR with specific primers designed from the tox region (positive for all of the P. sav. phaseolicola strains) and serotyping, as 71% of the P. sav. phaseolicola strains reacted as O-serogroup PHA1. Important intrapathovar variation was seen by genomic fingerprinting with REP and ERIC primers, as well as with RAPD primers (AE7 and AE10) and esterase profilings. While RAPD fingerprinting detected variability correlated with two race-associated evolutionary lines, REP, ERIC and esterase profiles revealed intrapathovar variation linked to some host origins, that separated the kudzu isolates, and the mungbean isolates, from the other P. sav. phaseolicola strains.

Pseudomonas lini sp. nov., a novel species from bulk and rhizospheric soils

The taxonomic position of eight fluorescent Pseudomonas strains isolated from bulk and rhizospheric soils, and from water was examined. These eight strains clustered in one phenon together with Pseudomomas mandelii (CFBP 4844T), but could still be differentiated from this type strain by four phenotypic features. The eight stains exhibited internal DNA-DNA hybridization values ranging from 60 to 100%, with deltaTm below 5 degrees C (3.9 and 4.3 degrees C) for the lowest values (60 and 66%). The percentages of hybridization with type or reference strains of other Pseudomonas species tested ranged from 12 to 60% (deltaTm = 5.5 degrees C), indicating that the eight isolates studied constituted a discrete DNA homology group. Comparison of the 16S rDNA sequence of the strain representing this group (CFBP 5737T) with the sequences of other strains belonging to the genus Pseudomonas revealed that strain CFBP 5737T was a member of this genus and that these bacteria did not cluster with any previously described species of the genus Pseudomonas. The eight isolates belonged to two siderovars different from those described so far. On the basis of the results of phenotypic, DNA-DNA and phylogenetic analyses, and of siderotyping, a new species, Pseudomonas lini sp. nov. (type strain CFBP 5737T) is proposed.

BOX-PCR-based identification of bacterial species belonging to Pseudomonas syringae: P. viridiflava group

The phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, with the aim of species identification. The numerical analysis of 119 nutritional characteristics did not show patterns that would help with identification. Regarding the genetic fingerprinting, the results of the present study supported the observation that BOX-PCR seems to be able to identify bacterial strains at species level. After numerical analyses of the bar-codes, all pathovars belonging to each one of the nine described genomospecies were clustered together at a distance of 0.72, and could be separated at genomic species level. Two P. syringae strains of unknown pathovars (CFBP 3650 and CFBP 3662) and the three P. syringae pv. actinidiae strains were grouped in two extra clusters and might eventually constitute two new species. This genomic species clustering was particularly evident for genomospecies 4, which gathered P. syringae pvs. atropurpurea, coronafaciens, garcae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance.