Rémi Chaussod

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.

Impact of agricultural practices on the size and activity of the microbial biomass in a long-term field experiment

The Dehérain long-term field experiment was initiated in 1875 to study the impact of fertilization on a wheat-sugarbeet rotation. In 1987, the rotation was stopped to be replaced by continuous maize. Crop residues were soil-incorporated and the mineral fertilization was doubled in some plots. The impact of those changes on the microbial biomass and activity are presented. In spring 1987, the soil was still in a steady-state condition corresponding to the rotation. The microbial biomass was correlated with total organic C and decreased in the order farmyard manure>mineral NPK>unfertilized control. Microbial specific respiratory activity was higher in the unfertilized treatments. The soil biomass was closely related to soil N plant uptake. In 1989, after 2 years of maize and crop residue incorporation, the steady-state condition corresponding to the previous agricultural practices disappeared. So did the relationship between the biomass and total organic C, and the soil N plant uptake. Biomass specific respiratory activity increased because of low efficiency in the use of maize residues by microbes under N stress.

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.

Chloroform fumigation technique as a means of determining the size of specialized soil microbial populations: application to pesticide-degrading microorganisms

A method is proposed for studying the dynamic behaviour of the soil microbial population involved in the degradation of 2,4-D. The method is based on in situ specific-labelling of that population following treatment of the soil with 14C-labelled herbicide and investigating the kinetics of the incorporation of radioactivity by the soil microflora in treated soil samples subjected to the chloroform-fumigation technique after varying periods of exposure. Non-degraded herbicide still present in the soil after fumigation did not affect the overall flush of CO2 and was not further broken down at a sufficient rate to appreciably contribute to 14CO2 evolution. The validity of the method to assess the soil biomass of the 2,4-D degrading population together with its time variations is discussed in relation to the position of the 14C on the pesticide molecule.

Decomposition de corps microbiens dans des sols fumiges au chloroforme: Effets du type de sol et de microorganisme

Five microbial species (Aspergillus flavus, Trichoderma viride, Streptomyces sp., Arthrobacter sp., Achromobacter liquefaciens) were cultivated in liquid media containing 14C-labelled glucose. The decomposition of these microorganisms was recorded in four different soils after chloroform fumigation by a technique related to that proposed by Jenkinson and Powlson, to determine the mineralization rate of microbial organic matter (Kc coefficient). Three treatments were used: untreated soil, fumigated soil alone and fumigated soil supplied with 14C-labelled cells. Total evolved CO2 and 14CO2 were measured after 7 and 14 days at 28°C. The labelled microorganisms enabled the calculation of mineralization rate Kc (Kc = mineralized microbial carbon/supplied microbial carbon). The extent of mineralization of labelled microbial carbon depended on the type of soil and on the microbial species. Statistical analysis of results at 7 days showed that 58% of the variance is taken in account by the soil effect and 32% by the microorganism effect. Between 35 and 49% of the supplied microbial C was mineralized in 7 days according to the soil type and the species of microorganism. Our results confirmed that the average value for Kc = 0.41 is acceptable, but Kc variability according to soil type must be considered. The priming effect on organic C and native microbial biomass mineralization, due to microbial carbon addition was obtained by comparison between the amount of non-labelled CO2-C produced by fumigated soils with or without added labelled microorganisms: this priming effect was generally negligible. These results indicate that the major portion of the error of microbial biomass measurement comes from the Kc estimation.

Typology of exogenous organic matters based on chemical and biochemical composition to predict potential nitrogen mineralization

Our aim was to develop a typology predicting potential N availability of exogenous organic matters (EOMs) in soil based on their chemical characteristics. A database of 273 EOMs was constructed including analytical data of biochemical fractionation, organic C and N, and results of N mineralization during incubation of soil-EOM mixtures in controlled conditions. Multiple factor analysis and hierarchical classification were performed to gather EOMs with similar composition and N mineralization behavior. A typology was then defined using composition criteria to predict potential N mineralization. Six classes of EOM potential N mineralization in soil were defined, from high potential N mineralization to risk of inducing N immobilization in soil after application. These classes were defined on the basis of EOM organic N content and soluble, cellulose-, and lignin-like fractions. A decision tree based on these variables was constructed in order to easily attribute any EOM to 1 of the 6 classes.

The argan tree (Argania spinosa) in Morocco: Propagation by seeds, cuttings and in-vitro techniques

In South-western Morocco, the argan tree (Argania spinosa) is basis of a traditional agroforestry system. However, this system is no longer at equilibrium and there is an urgent need to renovate it. The natural reproduction of the tree has become difficult, and most attempts of artificial regeneration have failed. We have assessed three different propagation methods: seedlings, cuttings, and in-vitro micro-propagation. Our experiments, involving several seed collections, showed that germination occurred easily when using young (< 12 months) and large seeds (nut weight > 3 g). Nuts with thin walls germinated better than nuts with thicker walls. Sterilization prevented microbial contamination and improved germination. Vegetative propagation of the argan tree by cuttings was tried using lignified cuttings collected from adult trees or young stems from managed stockplants (three years old). Rooting rate seems to be dependent on genotype, and the best results were obtained from young stems sterilized to avoid fungal contamination. The success of in-vitro micro-propagation is also highly genotype-dependant. We developed a modified medium enabling up to 80–95% rooting rate for some clones. However, other clones were still recalcitrant. Whatever the propagation method used, special attention must be paid to the architecture and growth of the root system.

The dynamics of soil bacterial community structure in response to yearly repeated agricultural copper treatments.

The annual dynamics of soil bacterial community structure, including early, dose-dependent and transient modifications, was observed consecutively at different levels of copper contamination (high: 48 kg Cu ha(-1), low: 16 kg Cu ha(-1)) repeated yearly over a three-year field experiment. Repeated low-level Cu contamination led to an increase in community stability to metal stress without a long-term shift in the population structure, whereas repeated high-level Cu contamination induced a novel and stable bacterial community structure. Furthermore, field experimentation highlighted that episodic climatic stress can modulate copper impact by enhancing community stability.

Ultrafiltration as a Means to Investigate Copper Resistance Mechanisms in Soil Bacteria

Copper is a trace element of major concern for agricultural soils. It exhibits a high toxicity against microorganisms and is widely introduced into soils as a component of pesticide treatments or urban wastes such as sewage sludges or refuse composts. In most French vineyards, “Bordeaux mixture” (copper sulfate) has been applied for more than a century, sometimes leading to copper concentrations in soils much higher than the threshold values (100 mg kg−1 in France). Microorganisms exposed to high concentrations of copper or other trace elements are known to develop resistance mechanisms and represent a suitable material for the study of such processes, both at a physiological and molecular level (Capasso et al., 1996).