Guy Soulas

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

ABSTRACT The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.

Dependence of accelerated degradation of atrazine on soil pH in French and Canadian soils

Abstract A series of agricultural soils varying in their atrazine treatment history were sampled from 12 sites in France and two sites in Canada. The soils varied widely with respect to soil chemical, physical and microbiological (total microbial biomass, kinetics of C and N mineralization) properties. Soils treated with as few as two successive atrazine field applications mineralized [U- ring - 14 C]atrazine significantly more rapidly in 35 d laboratory incubations than did soils which had never received atrazine. Longer treatment history tended to favour more rapid mineralization in the so-called “adapted” soils. Up to 80% of the initially applied 14 C-atrazine was mineralized at the end of the incubations in these adapted soils. Of the properties tested, soil pH was the most significantly related to atrazine mineralized. In soils with pH lower than 6.5, less than 25% of the initial 14 C-atrazine was mineralized even after repeated application in field conditions. Atrazine retention in soil did not influence its mineralization rate. Both hydroxylated and dealkylated atrazine metabolites were detected, but no clear pattern of metabolite production could be determined. Large amounts of bound residues were formed in soils that mineralized little atrazine.

Isolation and characterisation of Nocardioides sp. SP12, an atrazine-degrading bacterial strain possessing the gene trzN from bulk- and maize rhizosphere soil

We report the characterisation of Nocardioides sp. SP12, an atrazine-degrading bacteria isolated from atrazine-treated bulk- and maize rhizosphere soil. Based on 16S rDNA alignment, strain SP12 showed close phylogenic relationships with Nocardioides sp. C157 and Nocardioides simplex. Internal transcribed spacer (ITS) sequences of strain SP12 were longer than those of other Nocardioides sp. and present Ala- and Ile-tRNA unlike Actinomycetales. Nocardioides sp. SP12 presents a novel atrazine catabolic pathway combining trzN with atzB and atzC. Atrazine biodegradation ends in a metabolite that co-eluted in HPLC with cyanuric acid. This metabolite shows an absorption spectrum identical to that of cyanuric acid with a maximal absorption at 214.6 nm. The mass of the atrazine metabolite is in concordance with that of cyanuric acid according to mass spectrometry analysis. Quantitative PCR revealed that the ITS sequence of Nocardioides sp. SP12 was at a lower number than the one of trzN in atrazine-treated soil samples. It suggests that trzN could also be present in other atrazine degrading bacteria. The numbers of trzN and ITS sequences of Nocardioides sp. SP12 were higher in the maize rhizosphere than in bulk soil.

Accelerated mineralisation of atrazine in maize rhizosphere soil

Abstract. The mineralisation rate of atrazine measured in soil pre-treated with this herbicide, was significantly higher in the maize rhizosphere than in bulk soil. Maize rhizosphere was also shown to significantly increase microbial biomass C as compared with bulk soil. Ribosomal intergenic spacer analysis conducted on nucleic acids extracted directly from soil samples revealed that the structure of microbial communities observed in the rhizosphere was slightly different from that of bulk soil. The quantification of the relative amount of the gene atzC, which encodes an enzyme involved in atrazine mineralisation, was carried out on soil nucleic acids by using quantitative–competitive PCR assays. It revealed that atzC was present at a higher level in the rhizosphere than in bulk soil. In addition, the amount of atzC was transiently enhanced in both rhizosphere and bulk soils following atrazine treatment. These results suggest that the stimulation of atrazine mineralisation in the maize rhizosphere depends on the abundance of atrazine-degrading communities.

Estimation of atrazine-degrading genetic potential and activity in three French agricultural soils

The impact of organic amendment (sewage sludge or waste water) used to fertilize agricultural soils was estimated on the atrazine-degrading activity, the atrazine-degrading genetic potential and the bacterial community structure of soils continuously cropped with corn. Long-term application of organic amendment did not modify atrazine-mineralizing activity, which was found to essentially depend on the soil type. It also did not modify atrazine-degrading genetic potential estimated by quantitative PCR targeting atzA, B and C genes, which was shown to depend on soil type. The structure of soil bacterial community determined by RISA fingerprinting was significantly affected by organic amendment. These results showed that modification of the structure of soil bacterial community in response to organic amendment is not necessarily accompanied by a modification of atrazine-degrading genetic potential or activity. In addition, these results revealed that different soils showing similar atrazine-degrading genetic potentials may exhibit different atrazine-degrading activities.

Isolation and characterization of a stable 2,4-dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus, using chemostat culture

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway.

Diuron mineralisation in a Mediterranean vineyard soil: impact of moisture content and temperature

BACKGROUND The diuron-mineralising ability of the microbiota of a Mediterranean vineyard soil exposed each year to this herbicide was measured. The impact of soil moisture and temperature on this microbial activity was assessed. RESULTS The soil microbiota was shown to mineralise diuron. This mineralising activity was positively correlated with soil moisture content, being negligible at 5% and more than 30% at 20% soil moisture content. According to a double Gaussian model applied to fit the dataset, the optimum temperature/soil moisture conditions were 27.9 degrees C/19.3% for maximum mineralisation rate and 21.9 degrees C/18.3% for maximum percentage mineralisation. The impact of temperature and soil moisture content variations on diuron mineralisation was estimated. A simulated drought period had a suppressive effect on subsequent diuron mineralisation. This drought effect was more marked when higher temperatures were used to dry (40 degrees C versus 28 degrees C) or incubate (28 degrees C versus 20 degrees C) the soil. The diuron kinetic parameters measured after drought conditions were no longer in accordance with those estimated by the Gaussian model. CONCLUSION Although soil microbiota can adapt to diuron mineralisation, its activity is strongly dependent on climatic conditions. It suggests that diuron is not rapidly degraded under Mediterranean climate, and that arable Mediterranean soils are likely to accumulate diuron residues.

Isoproturon mineralization in an agricultural soil

The impact of soil moisture content and temperature on isoproturon (3-(4-isopropylphenyl)-1,1-dimethyl-urea [IPU]) mineralization activity was assessed on an agricultural soil regularly exposed to this herbicide. Mineralization of 14C-IPU was monitored on soil microcosms incubated at different temperatures (10°C, 20°C, 28°C) and soil moisture contents (9%, 12%, 15, 18%, 21%, 24%). An increase in temperature and/or soil moisture significantly enhanced the maximum rate and percentage of IPU mineralization while it decreased the lag time before mineralization. The maximum rate and percentage of IPU mineralization respectively ranged from 0.18% day−1 and 9% for the lowest temperature and soil moisture content pair (10°C–9%) to 1.51% day−1 and 27.1% for the highest pair (28°C–24%). Statistics revealed a cross interaction of temperature and soil moisture content on the maximum rate of IPU mineralization. The optimum conditions for IPU mineralization, estimated from the double Gaussian model, were 25.8°C and 24% soil moisture content. The influence of fluctuations in soil moisture content on IPU-mineralization was investigated by subjecting the soil microcosms to drought stress. When IPU was added at the end of the drought stress, it had no statistical effect on IPU mineralization. However, when it was added before the drought stress, two mineralization phases were observed: (1) one corresponding to the drought stress for which mineralization was low and (2) another one observed after restoration of soil moisture content characterized by higher mineralization rate. It can be concluded that climatic fluctuations affect the activity of IPU mineralizing microbial community, and may lead to an increase in IPU persistence.