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A PCR-based molecular tool was developed to estimate the diversity of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHS. A degenerate primer pair specific to catA sequences was designed by multiple alignment of known sequences coding a key intermediate of the beta-ketoadiapate pathway degrading catechol, namely catechol 1,2-dioxygenase. The specificity of this primer pair was assessed in 21 pure strains by PCR and sequencing. Comparison of the 16S rDNA and catA phylogenies revealed an absence of congruence between these two genes. The primer set was able to amplify catA sequences in DNA extracts from an industrial soil highly contaminated with heavy metals and polycyclic aromatic hydrocarbons (PAHs). RFLP screening of the catA library (95 clones) yielded 32 RFLP families. All of the 43 clone sequences obtained exhibited 86% identity on average to known CatA. Phylogenetic analysis revealed that these CatA sequences were related to Actinobacteria, alpha-, beta- and gamma-Proteobacteria phyla and confirmed the absence of congruence with 16S rDNA sequences, which implies horizontal gene transfer of the cat gene cluster between soil microbiota. Our results suggest that the diversity of the catA bacterial community is maintained in highly contaminated soil.

A PCR-based molecular tool was developed to estimate the diversity of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHS. A degenerate primer pair specific to catA sequences was designed by multiple alignment of known sequences coding a key intermediate of the beta-ketoadiapate pathway degrading catechol, namely catechol 1,2-dioxygenase. The specificity of this primer pair was assessed in 21 pure strains by PCR and sequencing. Comparison of the 16S rDNA and catA phylogenies revealed an absence of congruence between these two genes. The primer set was able to amplify catA sequences in DNA extracts from an industrial soil highly contaminated with heavy metals and polycyclic aromatic hydrocarbons (PAHs). RFLP screening of the catA library (95 clones) yielded 32 RFLP families. All of the 43 clone sequences obtained exhibited 86% identity on average to known CatA. Phylogenetic analysis revealed that these CatA sequences were related to Actinobacteria, alpha-, beta- and gamma-Proteobacteria phyla and confirmed the absence of congruence with 16S rDNA sequences, which implies horizontal gene transfer of the cat gene cluster between soil microbiota. Our results suggest that the diversity of the catA bacterial community is maintained in highly contaminated soil.

AbstractWe report the development of quantitative competitive (QC) PCR assays for quantifying the 16S, 18S ribosomal and atzC genes in nucleic acids directly extracted from soil. QC‐PCR assays were standardised, calibrated and evaluated with an experimental study aiming to evaluate the impact of atrazine application on soil microflora. Comparison of QC‐PCR 16S and 18S results with those of soil microbial biomass showed that, following atrazine application, the microbial biomass was not affected and that the amount of 16S rDNA gene representing ‘bacteria’ increased transitorily, while the amount of 18S rDNA gene representing fungi decreased in soil. In addition, comparison of atzC QC‐PCR results with those of atrazine mineralisation revealed that, in response to atrazine treatment, the amount of atzC gene increased transitorily in soil pre‐treated with atrazine, suggesting that accelerated atrazine biodegradation in soil could be due to a transient increase in the size of the atrazine mineralising community.© 2003 Society of Chemical Industry

AbstractWe report the development of quantitative competitive (QC) PCR assays for quantifying the 16S, 18S ribosomal and atzC genes in nucleic acids directly extracted from soil. QC‐PCR assays were standardised, calibrated and evaluated with an experimental study aiming to evaluate the impact of atrazine application on soil microflora. Comparison of QC‐PCR 16S and 18S results with those of soil microbial biomass showed that, following atrazine application, the microbial biomass was not affected and that the amount of 16S rDNA gene representing ‘bacteria’ increased transitorily, while the amount of 18S rDNA gene representing fungi decreased in soil. In addition, comparison of atzC QC‐PCR results with those of atrazine mineralisation revealed that, in response to atrazine treatment, the amount of atzC gene increased transitorily in soil pre‐treated with atrazine, suggesting that accelerated atrazine biodegradation in soil could be due to a transient increase in the size of the atrazine mineralising community.© 2003 Society of Chemical Industry

The insecticide chlordecone is a contaminant found in most of the banana plantations in the French West Indies. This study aims to search for fungal populations able to grow on it. An Andosol heavily contaminated with chlordecone, perfused for 1 year in a soil-charcoal system, was used to conduct enrichment cultures. A total of 103 fungal strains able to grow on chlordecone-mineral salt medium were isolated, purified, and deposited in the MIAE collection (Microorganismes d'Intérêt Agro-Environnemental, UMR Agroécologie, Institut National de la Recherche Agronomique, Dijon, France). Internal transcribed spacer sequencing revealed that all isolated strains belonged to the Ascomycota phylum and gathered in 11 genera: Metacordyceps, Cordyceps, Pochonia, Acremonium, Fusarium, Paecilomyces, Ophiocordyceps, Purpureocillium, Bionectria, Penicillium, and Aspergillus. Among predominant species, only one isolate, Fusarium oxysporum MIAE01197, was able to grow in a liquid culture medium that contained chlordecone as sole carbon source. Chlordecone increased F. oxysporum MIAE01197 growth rate, attesting for its tolerance to this organochlorine. Moreover, F. oxysporum MIAE01197 exhibited a higher EC50 value than the reference strain F. oxysporum MIAE00047. This further suggests its adaptation to chlordecone tolerance up to 29.2 mg l(-1). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that 40 % of chlordecone was dissipated in F. oxysporum MIAE01197 suspension culture. No chlordecone metabolite was detected by GC-MS. However, weak amount of (14)CO2 evolved from (14)C10-chlordecone and (14)C10-metabolites were observed. Sorption of (14)C10-chlordecone onto fungal biomass followed a linear relationship (r (2) = 0.99) suggesting that it may also account for chlordecone dissipation in F. oxysporum MIAE01197 culture.

The insecticide chlordecone is a contaminant found in most of the banana plantations in the French West Indies. This study aims to search for fungal populations able to grow on it. An Andosol heavily contaminated with chlordecone, perfused for 1 year in a soil-charcoal system, was used to conduct enrichment cultures. A total of 103 fungal strains able to grow on chlordecone-mineral salt medium were isolated, purified, and deposited in the MIAE collection (Microorganismes d'Intérêt Agro-Environnemental, UMR Agroécologie, Institut National de la Recherche Agronomique, Dijon, France). Internal transcribed spacer sequencing revealed that all isolated strains belonged to the Ascomycota phylum and gathered in 11 genera: Metacordyceps, Cordyceps, Pochonia, Acremonium, Fusarium, Paecilomyces, Ophiocordyceps, Purpureocillium, Bionectria, Penicillium, and Aspergillus. Among predominant species, only one isolate, Fusarium oxysporum MIAE01197, was able to grow in a liquid culture medium that contained chlordecone as sole carbon source. Chlordecone increased F. oxysporum MIAE01197 growth rate, attesting for its tolerance to this organochlorine. Moreover, F. oxysporum MIAE01197 exhibited a higher EC50 value than the reference strain F. oxysporum MIAE00047. This further suggests its adaptation to chlordecone tolerance up to 29.2 mg l(-1). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that 40 % of chlordecone was dissipated in F. oxysporum MIAE01197 suspension culture. No chlordecone metabolite was detected by GC-MS. However, weak amount of (14)CO2 evolved from (14)C10-chlordecone and (14)C10-metabolites were observed. Sorption of (14)C10-chlordecone onto fungal biomass followed a linear relationship (r (2) = 0.99) suggesting that it may also account for chlordecone dissipation in F. oxysporum MIAE01197 culture.

The dissolved organic matter (DOM) is the term used for organic components of natural origin present in the soil solution and is probably the most available C-source that primes microbial activity in subsoils. Contrasting effects of organic C components on pesticide degradation have been reported; however, most studies have used model organic compounds with compositions and concentrations which differ substantially from those found in the environment. Degradation of atrazine (AT) by Chelatobacter heintzii SalB was monitored in liquid batch assays in the absence or presence of well-defined model C compounds (glucose, gluconate and citrate) as model DOM (mDOM) or complex, less-defined, environmental DOM solutions (eDOM: isolated humic substances, soil and plant residue extracts) at environmentally relevant concentrations. Glucose significantly increased AT degradation rate by more than a factor of 8 at and above 2.5 mg C L( - 1). Optical density measurements showed that this stimulation is related to microbial growth. Gluconate and citrate had no effects unless at non-relevant concentrations (1,000 mg DOC L( - 1)) at which stimulations (gluconate) or inhibitions (citrate) were found. The effects of eDOM added at 10 mg DOC L( - 1) on AT degradation were generally small. The AT degradation time was reduced by factors 1.4-1.9 in the presence of humic acids and eDOM from soils amended with plant residues; however, no effects were found for fulvic acids or eDOM from a soil leachate solution or extracted from unamended peat or forest soil. In conclusion, DOM supplied as both mDOM and eDOM did not inhibit AT degradation at environmentally relevant concentrations, and stimulation can be found for selected DOM samples and this is partly related to its effect on growth.