Fig. 1. Phylogenetic tree inferred using the ARB software package by neighbour-joining analysis with Jukes–Cantor distance correction based on alignment of partial 16S rDNA sequences (900 bp), showing the phylogenetic affiliation of m-toluate tolerating isolates from oil-contaminated Galega orientalis rhizosphere. The scale bar represents 0.05 changes per nucleotide position. The number of isolates is indicated inside the brackets. Bacterial species either in bold or underlined in the figure includes catechol positive or catechol negative isolates able to degrade m-toluate, respectively.

Fig. 2. A UPGMA dendrogram of the isolates from oil-contaminated Galega orientalis rhizosphere based on 16S rDNA PCR-RFLP fingerprinting analysis with combined patterns of three restriction endonucleases: AluI, MspI and RsaI. The cophenetic correlation value of the dendrogram was 93.2% representing trustworthy the similarity matrix. E or –, catechol positive or negative, respectively; B, able to break down m-toluate as the sole C source.

Fig. 3. (GTG)₅-PCR fingerprinting of some isolates from oil-contaminated Galega orientalis rhizosphere. M, λSinI molecular marker. 1, Rhizobium galegae H1174; 2, Pseudomonas putida PaW85 H1828; 3, Rhodococcus erythropolis H2378; 4, H2616; 5, H2617; 6, Arthrobacter histidinolovorans H2379; 7, A. histidinolovorans H2381; 8, A. histidinolovorans H2383; 9, A. histidinolovorans H2372; 10, H1614; 11, P. oryzihabitans H2386; 12, H2387; 13, P. oryzihabitans H2388; 14, Bacillus thuringiensis H2389; 15, Nocardia calcarea H2390. Identification of the isolates is indicated in Table 1.

Fig. 4. UPGMA dendrograms of the gram-positive (a) and gram-negative (b) isolates from oil-contaminated Galega orientalis rhizosphere based on genomic fingerprinting by rep-PCR with (GTG)₅ primers. The reference strains Rhizobium galegae and Pseudomonas putida PaW85 (not isolated from the soils) were included in the dendrogram (b) for reference. The value of cophenetic correlation with gram-negative bacteria was 92.7% and with gram-positive bacteria 95.2% representing trustworthy the similarity matrix. E or –, catechol positive or negative, respectively; B, able to break down m-toluate as the sole C source.

Table 1.

Characteristics of the bacterial strains selected with m-toluate from the oil-contaminated rhizosphere of Galega orientalis

^a HAMBI, the Microbial Culture Collection at the Division of Microbiology, Department of Applied Chemistry and Microbiology, University of Helsinki, Finland; Acc. no, the number of the 16S rDNA sequence in DDBJ/EMBL/GenBank nucleotide sequence databases.
^b OS, oil soil, MS, m-toluate soil; GR, Galega orientalis inoculated with Rhizobium galegae; +P, Pseudomonas putida PaW85/pWWO inoculant in the peat layer.
^c DEF, defined medium DEF No 8, YEM, yeast extract-mannitol medium with streptomycin (500 μg/ml); SEA, soil extract agar; glu, glucose; man, mannitol; RE, rhizosphere extract, SE, soil extract.
^d Phylogenetic groups: α, β, γ, α-, β- and γ-subdivision of Proteobacteria, respectively, hGC and lGC, gram-positive bacteria with high and low G + C content, respectively.
^e Observation limit for m-toluate tolerance and utilization was 1 g/l, B, brown pigment, ND, no data.
^f Grouping of rhizosphere isolates according to their ability to degrade/tolerate m-toluate: 1, m-toluate degraders which were catechol-positive (C+), and used m-toluate excellently (a, 13 g/l), well (b, 8 g/l) or moderately (c, up to 4 g/l), 2, m-toluate degraders which were catechol-negative, and used m-toluate well (a, 7 g/l) or moderately (b, up to 5 g/l), 3, m-toluate tolerants which were catechol-negative, and tolerated m-toluate excellently (a, up to 13 g/l), well (b, up to 8 g/l) or moderately (c, up to 5 g/l).
^g Rhizosphere isolates were grouped according to the similarity (0.7) of their restriction patterns into 13 16S rDNA PCR-RFLP ribotypes in computer-assisted analysis combining restriction enzymes AluI, MspI and RsaI, A–C, subdivision was based on the visual detection of profiles of 16S rDNA PCR-RFLP analysis with ten restriction enzymes (AluI, MseI, MspI, RsaI, CfoI, DdeI, HaeIII, HinfI, MboI and TaqI).
^h Rhizosphere isolates were grouped according to the similarity (0.7) of their genomic fingerprinting patterns into 23 (GTG)₅-genotypes in computer-assisted analysis.
ⁱ 16S rDNA nucleotide sequences were identified by WWW/BLASTN 2.1.1 using DDBJ/EMBL/GenBank nucleotide sequence databases, NS, not sequenced.
^j Identification was based on partial sequencing of 16S rRNA genes.