Siderophore typing,a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads

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.     

Ectomycorrhizal symbiosis affects functional diversity of rhizosphere fluorescent pseudomonads

Here we characterized the effect of the ectomycorrhizal symbiosis on the genotypic and functional diversity of soil Pseudomonas fluorescens populations and analysed its possible consequences in terms of plant nutrition, development and health. Sixty strains of P. fluorescens were isolated from the bulk soil of a forest nursery, the ectomycorrhizosphere and the ectomycorrhizas of the Douglas fir (Pseudostuga menziesii) seedlings-Laccaria bicolor S238N. They were characterized in vitro with the following criteria: ARDRA, phosphate solubilization, siderophore, HCN and AIA production, genes of N2-fixation and antibiotic synthesis, in vitro confrontation with a range of phytopathogenic and ectomycorrhizal fungi, effect on the Douglas fir-L. bicolor symbiosis. For most of these criteria, we demonstrated that the ectomycorrhizosphere significantly structures the P. fluorescens populations and selects strains potentially beneficial to the symbiosis and to the plant. This prompts us to propose the ectomycorrhizal symbiosis as a true microbial complex where multitrophic interactions take place. Moreover it underlines the fact that this symbiosis has an indirect positive effect on plant growth, via its selective pressure on bacterial communities, in addition to its known direct positive effect.     

Phylogenetic diversity of fluorescent pseudomonads in agricultural soils from Korea

AIMS: To identify and compare the relative diversity and distribution of genotypes of culturable fluorescent pseudomonads from soils. METHODS AND RESULTS: Analysis of 160 isolates from seven soil samples using randomly amplified polymorphism DNA methods revealed 53 genotypes, which were subsequently identified by their 16S ribosomal DNA sequences. Phylogenetic analyses of the 53 genotypes along with 43 fluorescent pseudomonad type strains separated the genotypes into 10 distinct clusters that included two phylogenetic groups that were not represented by previously described type strains. CONCLUSIONS: The diversity of genotypes that was obtained from the soil samples was highly variable among the different soils and appeared to be associated with different soil management practices that also influence plant yields. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and phylogenetic analysis of these genotypes offers opportunities for study of phenotypic traits that may be associated within taxonomically related groups of fluorescent pseudomonad species and how these groups vary in relation to soil management practices.     

Evaluation of phenotypic and molecular typing techniques for determining diversity in Erwinia carotovora subspp. atroseptica

A number of phenotypic and molecular fingerprinting techniques, including physiological profiling (Biolog), restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC) and a phage typing system, were evaluated for their ability to differentiate between 60 strains of Erwinia carotovora ssp. atroseptica (Eca) from eight west European countries. These techniques were compared with other fingerprinting techniques, random amplified polymorphic DNA (RAPD) and Ouchterlony double diffusion (ODD), previously used to type this pathogen. Where possible, data were represented as dendrograms and groups/subgroups of strains identified. Simpson's index of diversity (Simpson's D) was used to compare groupings obtained with the different techniques which, with the exception of Biolog, gave values of 0.46 (RFLP), 0. 39 (ERIC), 0.83 (phage typing), 0.82 (RAPD) and 0.26 (ODD). Of the techniques tested, phage typing showed the highest level of diversity within Eca, and this technique will now form the basis of studies into the epidemiology of blackleg disease.     

A comparative evaluation of PCR ribotyping and ERIC PCR for determining the diversity of clinical Pseudomonas aeruginosa isolates

Two typing methods were evaluated, utilizing 62 clinical strains of Pseudomonas aeruginosa, to assess their usefulness as tools to study the bacterial diversity within this complex group. Genetic diversity was determined by PCR ribotyping and enterobacterial repetitive intergenic consensus (ERIC) PCR. By these methods, 9 and 36 genotypes were found, respectively. The result showed that ERIC PCR analysis is a more discriminatory method than PCR ribotyping analysis and traditional serotyping scheme. We suggest that maximum discrimination can be achievied by a combination of these methods.     

Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil

Background  

Phosphorus is an essential macronutrient for the growth of plants. However, in most soils a large portion of phosphorus becomes insoluble and therefore, unavailable to plants. Knowledge on biodiversity of phosphate-solubilizing fluorescent pseudomonads is essential to understand their ecological role and their utilization in sustainable agriculture.     

Influence of soil reaction on diversity and antifungal activity of fluorescent pseudomonads in crop rhizospheres

The diversity and antifungal activity of fluorescent pseudomonads isolated from rhizospheres of tea, gladiolus, carnation and black gram grown in acidic soils with similar texture and climatic conditions were studied. Biochemical characterisation including antibiotic resistance assay, RAPD and PCR-RFLP studies revealed a largely homogenous population. At soil pH (5.2), the isolates exhibited growth with varying levels of siderophore production, irrespective of crop rhizospheres. Two isolates with maximum chitinase production showed antagonism. The bacterial populations in general lacked the ability to produce deleterious traits such as cellulase, pectinase and hydrogen cyanide. However, increased pH levels beyond 5.2 caused reduction in metabolite production with reduced antifungal activity. The homogeneity of the bacterial population irrespective of crop rhizospheres together with decreased secondary metabolite production at higher pH levels reinstated the importance of soil over host plant in influencing rhizosphere populations. The studies also yielded acid tolerant chitinase producing antagonistic fluorescent pseudomonads.     

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.     

Molecular diversity in the bacterial community and the fluorescent pseudomonads group in natural and chlorobenzoate-stressed peat-forest soil

Bacterial community shifts in a soil microcosm spiked with 3-chlorobenzoate or 2,5-dichlorobenzoate were monitored. The V6-V8 variable regions of soil bacterial 16S rRNA and rDNA were amplified and separated by temperature gradient gel electrophoresis (TGGE) profiling. Culturing in the presence of 2.5 mM chlorinated benzoates suppressed 10 to 100 fold the total aerobic bacterial community but had no effect on the diversity within the group of fluorescent pseudomonads. In contrast, the uncultured bacterial community showed a decrease in the number of bands in the TGGE profiles of the chlorobenzoate-spiked treatments. Accordingly, the Shannon's diversity and equitability indices of these treatments reflected a decreasing trend in time. The approach allowed a direct assessment of community shifts upon contamination of soil.     

Comparative genetic diversity of the narG,nosZ, and 16S rRNA genes in fluorescent pseudomonads

The diversity of the membrane-bound nitrate reductase (narG) and nitrous oxide reductase (nosZ) genes in fluorescent pseudomonads isolated from soil and rhizosphere environments was characterized together with that of the 16S rRNA gene by a PCR-restriction fragment length polymorphism assay. Fragments of 1,008 bp and 1,433 bp were amplified via PCR with primers specific for the narG and nosZ genes, respectively. The presence of the narG and nosZ genes in the bacterial strains was confirmed by hybridization of the genomic DNA and the PCR products with the corresponding probes. The ability of the strains to either reduce nitrate or totally dissimilate nitrogen was assessed. Overall, there was a good correspondence between the reductase activities and the presence of the corresponding genes. Distribution in the different ribotypes of strains harboring both the narG and nosZ genes and of strains missing both genes suggests that these two groups of strains had different evolutionary histories. Both dissimilatory genes showed high polymorphism, with similarity indexes (Jaccard) of between 0.04 and 0.8, whereas those of the 16S rRNA gene only varied from 0.77 to 0.99. No correlation between the similarity indexes of 16S rRNA and dissimilatory genes was seen, suggesting that the evolution rates of ribosomal and functional genes differ. Pairwise comparison of similarity indexes of the narG and nosZ genes led to the delineation of two types of strains. Within the first type, the similarity indexes of both genes varied in the same range, suggesting that these two genes have followed a similar evolution. Within the second type of strain, the range of variations was higher for the nosZ than for the narG gene, suggesting that these genes have had a different evolutionary rate.     

Taxonomic heterogeneity of the collection strains of fluorescent pseudomonads

Typing of 13 strains of fluorescent pseudomonads from the Belarusian collection of nonpathogenic microorganisms (BIM) by ERIC-PCR and BOX-PCR revealed high level of genetic heterogeneity in bacteria, most of which have been previously identified as Pseudomonas fluorescens according to the classical scheme. Evaluation of the similarities of the 16S rRNA gene sequences and their phylogenetic analysis excluded affiliation of the bacteria under study within the same species and allowed them to be distributed within three relatively distant clusters of the genus Pseudomonas phylogenetic tree. While eight strains fell into the phylogenetic group of P. fluorescens, only one of them could be identified as P. fluorescens. Four strains clustered within the P. vancouverensis phylogenetic group, formed by new species, which have been described mainly according to the evaluation of genome relationships. One bacterium was related to a stable branch that did not contain any type strains of the known Pseudomonas spp. These results indicate taxonomic heterogeneity of collection strains of the fluorescent pseudomonads and demonstrate the necessity of identification of them considering the requirements of phylogenetic bacterial taxonomy.     

Effects of fluorescent pseudomonads on the potato blackleg syndrome

In four field trials in 1983 and 1984 potato tubers were inoculated by dipping them at planting in a suspension of Erwinia carotovora subsp. atroseptica and then treated with a powder formulation of two strains of fluorescent pseudomonads (B 10 and I 13) isolated from potatoes. lnoculation with E. carotovora increased blackleg and reduced emergence, plant growth, tuber size and weight compared with uninoculated controls. These effects were partially reversed by treatment of tubers with fluorescent pseudomonads which also reduced contamination by E. carotovora and the soft-rot potential of progeny tubers. In some trials a mixture of both pseudomonad isolates delayed the breakdown of the mother tuber although individual treatments did not.     

In situ characterization of mercury-resistant growth-promoting fluorescent pseudomonads

Pseudomonas fluorescens strains PRS9 and GRS1 (wild type) were made mercury resistant PRS9Hg(r) (147 microM HgCl2) and GRS1Hg(r) (55 microM HgCl2), respectively, in King's medium by enrichment selection and their in situ root colonization studies were carried out. Mercury resistant mutant of PRS9 was stable and resulted in significant increase in root and shoot fresh weight (P < 0.05). Both the mutants are positive for indoleacetic acid (IAA), 'P' solubilization and siderophore production. PRS9, potent 'P' solubilizer, exhibited higher 'P' solubilization as compared to GRS1. After 2 weeks of inoculation, the population level of wild type PRS9 and its mercury resistant mutants has increased (50 fold). Mercury resistance has no adverse effect on the growth promoting properties of mutants besides being comparable in its morphological and physiological properties with their wild type counterpart. Furthermore, mercury resistant character facilitates rhizospheric competition and thus helpful for establishment of growth promoting strains where metal ions are either limiting and/or present at toxic level.     

Genotypic and phenotypic diversity of PGPR fluorescent pseudomonads isolated from the rhizosphere of sugarcane (Saccharum officinarum L.)

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.