Corinne Leyval

Real-Time PCR quantification of PAH-ring hydroxylating dioxygenase (PAH-RHDα) genes from Gram positive and Gram negative bacteria in soil and sediment samples

Real-Time PCR based assays were developed to quantify Gram positive (GP) and Gram negative (GN) bacterial populations that are capable of degrading the polycyclic aromatic hydrocarbons (PAH) in soil and sediment samples with contrasting contamination levels. These specific and sensitive Real-Time PCR assays were based on the quantification of the copy number of the gene that encodes the alpha subunit of the PAH-ring hydroxylating dioxygenases (PAH-RHDalpha), involved in the initial step of the aerobic metabolism of PAH. The PAH-RHDalpha-GP primer set was designed against the different allele types present in the data base (narAa, phdA/pdoA2, nidA/pdoA1, nidA3/fadA1) common to the Gram positive PAH degraders such as Rhodococcus, Mycobacterium, Nocardioides and Terrabacter strains. The PAH-RHDalpha-GN primer set was designed against the genes (nahAc, nahA3, nagAc, ndoB, ndoC2, pahAc, pahA3, phnAc, phnA1, bphAc, bphA1, dntAc and arhA1) common to the Gram negative PAH degraders such as Pseudomonas, Ralstonia, Commamonas, Burkholderia, Sphingomonas, Alcaligenes, Polaromonas strains. The PCR clones for DNA extracted from soil and sediment samples using the designed primers showed 100% relatedness to the PAH-RHDalpha genes targeted. Deduced from highly sensitive Real-Time PCR quantification, the ratio of PAH-RHDalpha gene relative to the 16S rRNA gene copy number showed that the PAH-bacterial degraders could represent up to 1% of the total bacterial community in the PAH-contaminated sites. This ratio highlighted a positive correlation between the PAH-bacterial biodegradation potential and the PAH-contamination level in the environmental samples studied.

Arbuscular mycorrhizal contribution to heavy metal uptake by maize (Zea mays L.) in pot culture with contaminated soil

In two pot-culture experiments with maize in a silty loam (P2 soil) contaminated by atmospheric deposition from a metal smelter, root colonization with indigenous or introduced arbuscular mycorrhizal (AM) fungi and their influence on plant metal uptake (Cd, Zn, Cu, Pb, Mn) were investigated. Soil was γ-irradiated for the nonmycorrhizal control. In experiment 1, nonirradiated soil provided the mycorrhizal treatment, whereas in experiment 2 the irradiated soil was inoculated with spores of a fungal culture from P2 soil or a laboratory reference culture, Glomus mosseae. Light intensity was considerably higher in experiment 2 and resulted in a fourfold higher shoot and tenfold higher root biomass. Under the conditions of experiment 1, biomass was significantly higher and Cd, Cu, Zn and Mn concentrations significantly lower in the mycorrhizal plants than in the nonmycorrhizal plants, suggesting a protection against metal toxicity. In contrast, in experiment 2, biomass did not differ between treatments and only Cu root concentration was decreased with G. mosseae-inoculated plants, whereas Cu shoot concentration was significantly increased with the indigenous P2 fungal culture. The latter achieved a significantly higher root colonization than G. mosseae (31.7 and 19.1%, respectively) suggesting its higher metal tolerance. Zn shoot concentration was higher in both mycorrhizal treatments and Pb concentrations, particularly in the roots, also tended to increase with mycorrhizal colonization. Cd concentrations were not altered between treatments. Cu and Zn, but not Pb and Cd root-shoot translocation increased with mycorrhizal colonization. The results show that the influence of AM on plant metal uptake depends on plant growth conditions, on the fungal partner and on the metal, and cannot be generalized. It is suggested that metal-tolerant mycorrhizal inoculants might be considered for soil reclamation, since under adverse conditions AM may be more important for plant metal resistance. Under the optimized conditions of normal agricultural practice, however, AM colonization even may increase plant metal absorption from polluted soils.

Root colonization of maize by a Cd-sensitive and a Cd-tolerant Glomus mosseae and cadmium uptake in sand culture

A Cd-tolerant arbuscular mycorrhizal fungus, Glomus mosseae, isolated from a polluted soil (P2 culture), was compared with a Cd-sensitive reference Glomus mosseae (Gm) for its capacity to colonize maize (Zea mays L.) roots and to affect plant growth and Cd uptake in sand culture at increasing exposure to Cd added with the nutrient solution (0, 0.1, 1, 5 and 10 mg L-1). After eight weeks, mycorrhizal colonization by P2 culture was relatively high (50% of the control without Cd added) up to 5 mg L-1 Cd, whereas colonization by Gm was almost totally suppressed at that Cd level. However, even mycorrhizal colonization by the Cd-tolerant P2 culture appeared more sensitive to Cd than plant growth and was completely suppressed at 10 mg L-1 Cd. AM colonization did not alleviate the negative effect of Cd on plant growth compared to the non-mycorrhizal treatment. On the contrary, at the 5 mg L-1 Cd level non-mycorrhizal plants were greater than mycorrhizal plants with lower Cd concentration.

Time-course of heavy metal uptake in maize and clover as affected by root density and different mycorrhizal inoculation regimes

Abstract. A pot experiment was conducted to test the effect of three microbial regimes on the time course of heavy metal uptake in clover and maize from an industrially polluted soil. The three treatments included: (1) an intact flora of bacteria and fungi, including indigenous arbuscular mycorrhizal (AM) fungi together with soil microfauna; (2) the indigenous bacterial/fungal flora except AM fungi, reintroduced into sterilized soil; or (3) the same bacterial/fungal flora plus an AM fungus. For the final harvest, two pot sizes were included to assess the effect of root density. Plant uptake of P and heavy metals varied according to plant species, harvest time and soil treatment. For both plant species, shoot concentration of Zn, Cd and Cu decreased and Ni increased with plant age. Plants growing in sterilized soil with reintroduced AM fungi generally grew better, but contained higher concentrations of heavy metals than those colonized by indigenous AM fungi. Plants with mycorrhiza frequently contained more P, Zn, Cd, Cu and Pb in roots and shoots compared to nonmycorrhizal plants. Elevated root/shoot concentration ratios of P and metals indicate a sequestration of metal phosphates in mycorrhizal roots. Mycorrhizal performance was influenced by root density. At low root densities, metal concentrations in mycorrhizal plants were reduced, whereas it had no effect at high root densities when the entire soil volume was efficiently exploited by roots. We conclude that root density data are essential for interpretations of the influence of AM on metal uptake in plants.

Fate of polycyclic aromatic hydrocarbons (PAH) in the rhizosphere and mycorrhizosphere of ryegrass.

Polycyclic aromatic hydrocarbons (PAH) can be degraded in the rhizosphere but may also interact with vegetation by accumulation in plant tissues or adsorption on root surface. Previous studies have shown that arbuscular mycorrhizal (AM) fungi contribute to the establishment and maintenance of plants in a PAH contaminated soil. We investigated the fate of PAH in the rhizosphere and mycorrhizosphere including biodegradation, uptake and adsorption. Experiments were conducted with ryegrass inoculated or not with Glomus mosseae P2 (BEG 69) and cultivated in pots filled with soil spiked with 5 g kg−1 of anthracene or with 1 g kg−1 of a mixture of 8 PAH in a growth chamber. PAH were extracted from root surfaces, root and shoot tissue and rhizosphere soil and were analysed by GC-MS. In both experiments, 0.006 – 0.11‰ of the initial extractable PAH concentration were adsorbed to roots, 0.003 – 0.16‰ were found in root tissue, 0.001‰ in shoot tissue and 36 – 66% were dissipated, suggesting that the major part of PAH dissipation in rhizosphere soil was due to biodegradation or biotransformation. With mycorrhizal plants, anthracene and PAH were less adsorbed to roots and shoot tissue concentrations were lower than with non mycorrhizal plants, which could contribute to explain the beneficial effect of AM fungi on plant survival in PAH contaminated soils.

Differential tolerance to Cd and Zn of arbuscular mycorrhizal (AM) fungal spores isolated from heavy metal-polluted and unpolluted soils

Spores of arbuscular mycorrhizal fungi were isolated from two soils of field trials at INRA-Bordeaux (France) polluted by long-term application of a zinc-polluted sewage sludge (S2 soil) or treated with cadmium nitrate (Cd40 soil) and from corresponding unpolluted soils (F and Cd0 soils). These AM fungi were tested for their tolerance to Cd and Zn added as salt solutions with increasing concentrations (0 to 10 mg L−1) in a simple spore germination device.According to preliminary identification the predominant species in S2 and F cultures was Glomus mosseae, whereas Cd40 and Cd0 cultures contained a mixture of at least G. mosseae and G. etunicatum. Germination of Cd40 spores was more tolerant to Cd and Zn than for Cd0 spores, with EC50 values of 73 and 158 μmol L−1 added Cd and Zn corresponding to approximately 10 and 13 μmol L−1 remaining in solution in the device. The S2 spores from the sludge contaminated soil were more tolerant to Zn (EC50=87 μmol L−1), but not to Cd (EC50=7.5 μmol L−1), than the spores from the farmyard manure-treated F soil (EC50=38 and 8.8 μmol L−1, respectively). Thus, S2 culture exhibited a specific tolerance to Zn, which was lower than the unspecific tolerance of Cd40 culture to both Cd and Zn, despite the much higher Zn availability in S2 soil.These results indicate that AM fungi from different soils may differ in their metal susceptibility and that both metal specific and unspecific tolerance mechanisms may be selected in metal polluted soils.

Rhizodeposition and net release of soluble organic compounds by pine and beech seedlings inoculated with rhizobacteria and ectomycorrhizal fungi

SummaryA lysimetric experiment was performed in a greenhouse to evalute root deposition and net release of soluble organic compounds after 1 and 2 years from pine and beech seedlings inoculated with an ectomycorrhizal fungus (Laccaria laccata) and/or rhizobacteria (Agrobacterium radiobacter for beech and Agrobacterium sp. for pine). Total C compounds released in the rhizosphere of both plants increased after inoculation with the bacteria or ectomycorrhizal fungus. The rhizobacteria increased root and plant growth and rhizodeposition, but the mycorrhizal fungi appeared to increase only root deposition. Soluble C compounds, collected after 2 years, represented only 0.1–0.3% of the total C compounds released into the rhizosphere, and were modified by inoculation with the microorganisms. After inoculation with the bacteria, levels of sugars and amino acids decreased in pine and beech rhizospheres, whereas organic acids increased, especially in the pine rhizosphere. In the rhizosphere of mycorrhizal beeches, sugar and amino acids increased, and organic acids differed from those released from non-mycorrhizal beeches. In the mycorrhizal pine rhizosphere, however, all compounds decreased. Following dual inoculations, mycorrhizal colonization increased, no effect on plant growth was observed, and virtually no organic acids were detected.

Solubility of metals in an anoxic sediment during prolonged aeration.

This work was conducted to study the evolution of the solubility of selected metals during the aeration of an anoxic sediment. Batch experiments were carried out for 76 days with a metal-polluted dredged sediment. The pH, Eh and concentration of Al, Cu, Fe, Hg, Pb and Zn were periodically recorded. Results showed that during the early stages of aeration, the solubility of metals increased rapidly but was then followed by fast re-adsorption. As a consequence, after 14 days most of the metals excepted Cu and Zn were present at low or undetectable concentrations in solution. Re-adsorption of Zn was observed to be much slower during the first two weeks, whereas solubilisation of Cu increased gradually during months after land disposal. According to speciation calculations, Cu solubilisation was in part due to complexation in solution by carbonates. In the case of Hg, although complexation by dissolved organic matter (DOM) could be expected, re-adsorption was the dominant process. However, more knowledge about the behaviour of the DOM present in anoxic sediments is needed in order to make more quantitative statements about the mobility of heavy metals contained in dredged material.

Spatial Distribution of Bacterial Communities and Phenanthrene Degradation in the Rhizosphere of Lolium perenne L.

ABSTRACT Rhizodegradation of organic pollutants, such as polycyclic aromatic hydrocarbons, is based on the effect of root-produced compounds, known as exudates. These exudates constitute an important and constant carbon source that selects microbial populations in the plant rhizosphere, modifying global as well as specific microbial activities. We conducted an experiment in two-compartment devices to show the selection of bacterial communities by root exudates and phenanthrene as a function of distance to roots. Using direct DNA extraction, PCR amplification, and thermal gradient gel electrophoresis screening, bacterial population profiles were analyzed in parallel to bacterial counts and quantification of phenanthrene biodegradation in three layers (0 to 3, 3 to 6, and 6 to 9 mm from root mat) of unplanted-polluted (phenanthrene), planted-polluted, and planted-unpolluted treatments. Bacterial community differed as a function of the distance to roots, in both the presence and the absence of phenanthrene. In the planted and polluted treatment, biodegradation rates showed a strong gradient with higher values near the roots. In the nonplanted treatment, bacterial communities were comparable in the three layers and phenanthrene biodegradation was high. Surprisingly, no biodegradation was detected in the section of planted polluted treatment farthest from the roots, where the bacterial community structure was similar to those of the nonplanted treatment. We conclude that root exudates and phenanthrene induce modifications of bacterial communities in polluted environments and spatially modify the activity of degrading bacteria.

Bioavailability of heavy metals and arbuscular mycorrhiza in a sewage-sludge-amended sandy soil

Abstract The bioavailability of metals (Cd, Ni, Zn, Cu, Pb and Mn) and abundance of arbuscular mycorrhiza were studied in a long-term sewage-sludge field trial on an acid sandy soil, at INRA-Bordeaux, France. Zn Mn-(E1) and Cd Ni-(E2) contaiminated sludge had been applied at two rates (10t DM ha−1 y−1 and 100 t DM ha−1 2y−1) for 18 and 5 y, respectively. Inorganic fertilizer and farm yard manure treatments served as unpolluted controls. Soil extraction with EDTANH4OAc and Ca(NO3)2 and plant (Zea mays L.) uptake demonstrated an unusually high Zn (E1), Cd and Ni (E2) availability in the sludge-amended plots. The spore density of arbuscular mycorrhizal fungi ranged from 16 to 67 spores 50 g−1 dry soil, and root colonization between 0–33%. No relationship between mycorrhizal abundance and degree of metal exposure in soil or inside plant roots could be established, but root colonization across the different treatments correlated well with plant P status. The results suggest a better tolerance of the indigenous population of arbuscular mycorrhizal fungi to elevated metal than to high P concentrations.