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In iron-deficient conditions, most bacteria and fungi are known to release siderophores, iron-chelating compounds. Most plants do not produce siderophores, but seem to use microbial siderophores as iron sources. Although ectomycorrhizal fungi have been found to release siderophores in pure culture, little research has addressed production by mycorrhizas and the consequences for plant iron nutrition. The objectives of this study were to determine the effect of an ectomycorrhizal fungus [Pisolithus tinctorius (Pers.) Coker and Couch] on the utilization of siderophore (ferrioxamine B) by slash pine (Pinus elliottii Engelm.) roots grown under iron-deficient or iron-sufficient conditions. Experiments were conducted with excised roots and whole seedlings. Uptake of 55 Fe from ferrioxamine B was lower by mycorrhizal roots than non-mycorrhizal roots. Growth under iron-deficient conditions had little effect on iron uptake by non-mycorrhizal roots but increased the uptake by mycorrhizal roots. Uptake of iron from a non-purified siderophore isolated from a pure culture of P. tinctorius was also lower by mycorrhizal roots. The uptake of iron was not dependent on the pH of the uptake solution. Differential responses could be attributed to different mechanisms of iron uptake between fungal cells and root cells. However, the higher iron content of mycorrhizal roots may indicate a negative feedback effect on uptake.

Dark septate endophytes (DSEs) are widely distributed worldwide and can promote plant growth. Therefore, they are considered potentially important plant allies, especially in stressful environments. Previous studies have reported that DSEs cohabit roots with other microorganisms such as ectomycorrhizal (ECM), endophytic and pathogenic fungi/oomycetes. However, interactions between different DSE species have not yet been reported, and studies on the interactions between DSEs and other fungi are scarce. Using a simple and reproducible pairwise growth assay in vitro, we studied the synergistic/antagonistic interactions between eight DSEs, two ECM fungi and three root pathogens. Most of the DSE/DSE outcomes were neutral. Interestingly, we identified several DSE strains acting in synergy with other strains, as well as strains that could potentially act as biocontrol agents. Notably, three metal-tolerant DSE strains, namely, Cadophora sp., Leptodontidium sp. and Phialophora mustea, could decrease the growth of the root phytopathogens Pythium intermedium, Phytophthora citricola and Heterobasidion annosum. The present data are discussed in the general context of the use of fungal consortia as inocula in the tree-based phytomanagement of marginal lands.

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International audience; Industrial wasteland soils with aged PAH and heavy metal contaminations are environments where pollutant toxicity has been maintained for decades. Although the communities may be well adapted to the presence of stressors, knowledge about microbial diversity in such soils is scarce. Soil microbial community dynamics can be driven by the presence of plants, but the impact of plant development on selection or diversification of microorganisms in these soils has not been established yet. To test these hypotheses, aged-contaminated soil samples from a field trial were collected. Plots planted with alfalfa were compared to bare soil plots, and bacterial and fungal diversity and abundance were assessed after 2 and 6 years. Using pyrosequencing of 16S rRNA gene and ITS amplicons, we showed that the bacterial community was dominated by Proteobacteria, Actinobacteria, and Bacteroidetes and was characterized by low Acidobacteria abundance, while the fungal community was mainly represented by members of the Ascomycota. The short-term toxic impact of pollutants usually reduces the microbial diversity, yet in our samples bacterial and fungal species richness and diversity was high suggesting that the community structure and diversity adapted to the contaminated soil over decades. The presence of plants induced higher bacterial and fungal diversity than in bare soil. It also increased the relative abundance of bacterial members of the Actinomycetales, Rhizobiales, and Xanthomonadales orders and of most fungal orders. Multivariate analysis showed correlations between microbial community structure and heavy metal and PAH concentrations over time, but also with edaphic parameters (C/N, pH, phosphorus, and nitrogen concentrations).

Dark septate endophytes (DSEs) are abundant in stressful environments, including trace element (TE)-enriched soils. However, knowledge about the effects of DSEs on plant growth in such soils is poor compared to the well-known mycorrhizal fungi. The aim of this work was to evaluate the effects of three DSE strains isolated from TE-contaminated soils on the growth and mineral nutrition of Betula pendula and Populus tremula x alba grown on two contrasting TE-polluted soils. The three DSEs evenly colonized the two plant species in both soils. Nevertheless, plant responses to DSE inoculation varied from neutral to beneficial depending on soil properties. Depending on fungal strain and plant species, different factors seemed to contribute to plant growth promotion. Phialophora mustea Pr27 and Leptodontidium Pr30 decreased lipid peroxidation in birch shoots. Chlorophyll, K, and P concentrations increased in the shoots of Leptodontidium Pr30-inoculated trees, whereas Cd concentration decreased in Cadophora Fe06-inoculated birch. The absence of a general DSE-mediated plant growth-promoting behavior could represent a limiting factor for a generic use of DSEs in the tree-based phytomanagement of TE-contaminated soils. Our results suggest that the selection of strains adapted to particular edaphic conditions should not be overlooked within the framework of phytomanagement.

International audience; Dark septate endophytes (DSEs) are melanin-enriched ascomycetous fungi that are abundant in stressed environments. However, little is known about their physiology and metabolism, and DSE genes have not been functionally characterized yet. Therefore developing molecular genetic tools to investigate the biological function of genes of interest in DSEs is of major significance. We investigated Agrobacterium tumefaciens-mediated transformation (ATMT) efficiency in eight DSE strains belonging to Cadophora sp., Cadophora malorum, Leptodontidium sp., Phialophora mustea, and Cladosporium cladosporioides. ATMT efficiency was DSE-dependent and ranged from 0.6% to 125 %. The transformants were mitotically stable and expressed the gfp marker. The patterns of transformant appearance as a function of time were diverse and also strain-dependent. We further focused on the effect of mycelium ageing on ATMT efficiency. Leptodontidium sp. Me07, Leptodontidium sp. Pr30, and C. cladosporioides CBS 101367 were significantly more transformed using fifteen-day-old mycelium (44.5, 6.9, and 1.1%, respectively) as compared to two-day-old mycelium (121, 28.7, and 25.1, respectively), whereas P. mustea Pr29 was more transformed using young mycelium (21.5% compared to 5.3% for the old mycelium). Finally, we focused on the effect of melanin content on ATMT efficiency. Melanin content in mycelium ranged from 0.9 to 3.2 mg per gram of dry weight. Tricyclazole, an inhibitor of melanin biosynthesis, negatively modulated melanin content, while copper, an inducer of melanin biosynthesis, positively modulated them. However there was no correlation between hyphal melanin content and ATMT efficiency.

Numerous studies address the distribution and the diversity of dark septate endophytes (DSEs) in the literature, but little is known about their ecological role and their effect on host plants, especially in metal-polluted soils. Seven DSE strains belonging to Cadophora, Leptodontidium, Phialophora and Phialocephala were isolated from roots of poplar trees from metal-polluted sites. All strains developed on a wide range of carbohydrates, including cell-wall-related compounds. The strains evenly colonized birch, eucalyptus and ryegrass roots in re-synthesis experiments. Root and shoot growth promotion was observed and was both plant and strain dependent. Two Phialophora and Leptodontidium strains particularly improved plant growth. However, there was no correlation between the level of root colonization by DSEs and the intensity of growth promotion. All strains produced auxin and six also stimulated plant growth through the release of volatile organic compounds (VOCs). SPME-GC/MS analyses revealed four major VOCs emitted by Cadophora and Leptodontidium The strains exhibited growth at high concentrations of several metals. The ability of metal-resistant DSE strains to produce both soluble and volatile compounds for plant growth promotion indicates interesting microbial resources with high potential to support sustainable production of bioenergy crops within the context of the phytomanagement of metal-contaminated sites.

The objectives of this study were to determine whether the invasive plant Amaranthus viridis influenced soil microbial and chemical properties and to assess the consequences of these modifications on native plant growth. The experiment was conducted in Senegal at two sites: one invaded by A. viridis and the other covered by other plant species. Soil nutrient contents as well as microbial community density, diversity and functions were measured. Additionally, five sahelian Acacia species were grown in (1) soil disinfected or not collected from both sites, (2) uninvaded soil exposed to an A. viridis plant aqueous extract and (3) soil collected from invaded and uninvaded sites and inoculated or not with the arbuscular mycorrhizal (AM) fungus Glomus intraradices. The results showed that the invasion of A. viridis increased soil nutrient availability, bacterial abundance and microbial activities. In contrast, AM fungi and rhizobial development and the growth of Acacia species were severely reduced in A. viridis-invaded soil. Amaranthus viridis aqueous extract also exhibited an inhibitory effect on rhizobial growth, indicating an antibacterial activity of this plant extract. However, the inoculation of G. intraradices was highly beneficial to the growth and nodulation of Acacia species. These results highlight the role of AM symbiosis in the processes involved in plant coexistence and in ecosystem management programs that target preservation of native plant diversity.

Some plants are more mycorrhizal than others and mycorrhizal colonisation of plants in extreme environments is frequently additionally reduced due to decreased spore density and/or diversity and therefore frequently overlooked. We analysed two plant species from both metal polluted and saline enriched soils with differing mycorrhizal colonisation levels/status using classical and molecular methods. The selected plant species were Sesleria caerulea (L.) Ard. and Thlaspi praecox Wulfen from a metal polluted site, and Limonium angustifolium (Tausch) Degen [Statice serotina Rchb., L. vulgare Mill. subsp. Serotinum (Rchb.) Gams] and Salicornia europaea L. from the Secovlje salterns in Slovenia. Despite the high mycorrhizal frequencies (F%) observed, the presence of arbuscules (A%) was at best low in S. caerulea and T. praecox, and undetectable in L. angustifolium and S. europaea. Temporal temperature gradient gel electrophoresis (TTGE) was applied to field-collected samples from both burdened environments and proved to be an effective technique for rapid profiling and identification of arbuscular mycorrhizal fungi (AMF). Sequencing and phylogenetic analysis confirmed the association of AMF of the genus Glomus with roots of all four plant species. This is the first report on the identification and profiling of Glomeromycota in the field-collected Cd/Zn metal hyperaccumulator T. praecox growing at a highly metal polluted site, as well as in L. angustifolium and S. europaea collected in a saline environment. The identification of AMF from both ecosystems only partially resembles previous identifications on the basis of spores.

International audience; The high organic pollutant concentration of aged polycyclic aromatic hydrocarbon (PAH)-contaminated wasteland soils is highly recalcitrant to biodegradation due to its very low bioavailability. In such soils, the microbial community is well adapted to the pollution, but the microbial activity is limited by nutrient availability. Management strategies could be applied to modify the soil microbial functioning as well as the PAH contamination through various amendment types. The impact of amendment with clay minerals (montmorillonite), wood sawdust and organic matter plant roots on microbial community structure was investigated on two aged PAH-contaminated soils both in laboratory and 1-year on-site pot experiments. Total PAH content (sum of 16 PAHs of the US-EPA list) and polar polycyclic aromatic compounds (pPAC) were monitored as well as the available PAH fraction using the Tenax method. The bacterial and fungal community structures were monitored using fingerprinting thermal gradient gel electrophoresis (TTGE) method. The abundance of bacteria (16S rRNA genes), fungi (18S rRNA genes) and PAH degraders (PAH-ring hydroxylating dioxygenase and catechol dioxygenase genes) was followed through qPCR assays. Although the treatments did not modify the total and available PAH content, the microbial community density, structure and the PAH degradation potential changed when fresh organic matter was provided as sawdust and under rhizosphere influence, while the clay mineral only increased the percentage of catechol-1,2-dioxygenase genes. The abundance of bacteria and fungi and the percentage of fungi relative to bacteria were enhanced in soil samples supplemented with wood sawdust and in the plant rhizospheric soils. Two distinct fungal populations developed in the two soils supplemented with sawdust, i.e. fungi related to Chaetomium and Neurospora genera and Brachyconidiellopsis and Pseudallescheria genera, in H and NM soils respectively. Wood sawdust amendment favoured the development of PAH-degrading bacteria holding Gram-negative PAH-ring hydroxylating dioxygenase, catechol-1,2-dioxygenase and catechol-2,3-dioxygenase genes. Regarding the total community structure, bacteria closely related to Thiobacillus (beta-Proteobacteria) and Steroidobacter (gamma-Proteobacteria) genera were favoured by wood sawdust amendment. In both soils, plant rhizospheres induced the development of fungi belonging to Ascomycota and related to Alternaria and Fusarium genera. Bacteria closely related to Luteolibacter (Verrucomicrobia) and Microbacterium (Actinobacteria) were favoured in alfalfa and ryegrass rhizosphere.

Biodegradation of polycyclic aromatic hydrocarbons (PAH) in soil is mainly performed by endogenous bacteria. The density and activity of soil bacteria are usually increased in the rhizosphere. A compartmentalized device was used to follow biodegradation of phenanthrene and bacterial community structure as a function of distance from roots. Isolation of total DNA and RNA, followed by PCR-TTGE and RT-PCR-TTGE on a 16S rDNA partial sequence, was performed to describe the structure of bacterial community and of active species, respectively. After 4 weeks, active species profiles in the immediate vicinity of roots were the same as in non-planted treatment, with similar biodegradation efficiency (approx. 60%) whereas community structure clearly indicated that bacterial populations were different. On the opposite, at a furthest distance (6–9 mm) from the roots where biodegradation was lower, bacterial community structure was similar to the non-planted treatment whereas active species differed. A specie activity factor (Saf) was calculated for five relevant species to follow their transcriptional state in the rhizosphere and showed spatial variations as a function of species and distance from roots. We conclude that depending on distance from roots, the rhizosphere selected different bacterial communities, and different active species within these communities, resulting in different degradation values. Potential biases of molecular protocols used in this study are discussed as well as their relevance to describe the bacterial component of the rhizosphere involved in PAH biodegradation.

Interactions between the plant and its microbial communities in the rhizosphere control microbial polycyclic aromatic hydrocarbons (PAH) biodegradation processes. Arbuscular mycorrhizal (AM) fungi can influence plant survival and PAH degradation in polluted soil. This work was aimed at studying the contribution of the mycorrhizosphere to PAH biodegradation in the presence of ryegrass (Lolium perenne L., cv. Barclay) inoculated with Glomus mosseae (BEG 69) by taking into account the structure and activity of bacterial communities, PAH degrading culturable bacteria as a function of the distance from roots. Ryegrass was grown in compartmentalized systems designed to harvest successive sections of rhizosphere in lateral compartments polluted or not with phenanthrene (PHE). Colonization of roots by G. mosseae (BEG 69) modified the structure and density of bacterial populations in the mycorrhizosphere, compared to the rhizosphere of non-mycorrhizal plants. G. mosseae increased the density of culturable heterotrophic and PAH degrading bacteria beyond the immediate rhizosphere in the presence of PHE, and increased the density of PAH degraders in the absence of the pollutant. Biodegradation was not significantly increased in the mycorrhizosphere, compared to control non-mycorrhizal plants, where PHE biodegradation already reached 92% after 6 weeks. However, dioxygenase transcriptional activity was found to be higher in the immediate mycorrhizosphere in the presence of G. mosseae (BEG 69).

Bioremediation technologies of Polycyclic Aromatic Hydrocarbons (PAH) are often limited by the recalcitrance to biodegradation of high molecular weight (HMW) PAH. Rhizosphere is known to increase the biodegradation of PAH but little is known about the biodegradability of these HMW compounds by rhizosphere bacteria. This study compared the effects of a 3 and a 5-ring PAH, phenanthrene (PHE) and dibenzo[a,h]anthracene (dBA) respectively, on the composition of bacterial community, the bacterial density and the biodegradation activity. Compartmentalized devices were designed to harvest three consecutive sections of the rhizosphere. Rhizosphere and non-rhizosphere compartments were filled with PHE or dBA spiked or unspiked sand and inoculated with a soil bacterial inoculum. Different bacterial communities and degradation values were found 5 weeks after spiking with PHE (41-76% biodegradation) and dBA (12-51% biodegradation). In sections closer to the root surface, bacterial populations differed as a function of the distance to roots and the PAH added, whereas in further rhizosphere sections, communities were closer to those of the non-planted treatments. Biodegradation of PHE was also a function of the distance to roots, and decreased from 76 to 42% within 9 mm from the roots. However, biodegradation of dBA was significantly higher in the middle section (3-6 mm from roots) than the others. Rhizosphere degradation of PAH varies with the nature of the PAH, and C fluxes from roots could limit the degradation of dBA.

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.

Rhizodegradation of recalcitrant organic pollutants, such as polycyclic aromatic hydrocarbons (PAH), may benefit from the major role that root exudates have on rhizospheric microbial processes. We investigated the influence of the proximity to ryegrass (Lolium perenne L.) roots on microbial populations and their biodegradation of phenanthrene (PHE) using compartmented pots. PAH degrading bacteria, total heterotrophic bacteria and PHE biodegradation were quantified in three consecutive sections at different distance (0–3, 3–6, 6–9 mm) from a mat of actively exuding roots. A bacterial gradient was observed with higher numbers of heterotrophs and PAH degrading bacteria closest to the roots. In parallel, a PHE biodegradation gradient was evident in the presence of roots. The biodegradation reached 86%, 48% and 36% of initially added PHE, respectively, in the layers 0–3 mm, 3–6 and 6–9 mm from the roots. The biodegradation rate was similar throughout the three layers of the non planted control. The present experimental system seems well suited for spatial and dynamic studies of PAH rhizoremediation.

Phytoremediation as a means of cleaning up polluted soils has gained popularity during the last decade due to its convenience and low costs of installation and maintenance. When the target pollutant is biodegradable, this technology exploits the stimulating effect that roots have on microbial processes and physical/chemical modifications in the rhizosphere. Among the microorganisms that affect rhizosphere processes, symbiotic fungi forming mycorrhizas induce a series of changes in plant physiology, nutrient availability and microbial composition that may determine the outcome of a phytoremediation attempt. Beyond the rhizosphere, mycorrhizal hyphae act as the roots of the roots, and may thus extend the rhizosphere into the bulk soil by creating a new interface of soil-plant interactions; the hyphosphere. We here discuss some of the recent results on phytoremediation of organic pollutants with emphasis on processes in the mycorrhizosphere, and highlight future research priorities.

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.

It is known that plant rhizospheres can enhance the dissipation of polycyclic aromatic hydrocarbons (PAHs), but the mechanisms involved are not fully understood. The contribution of root symbiotic micro-organisms such as arbuscular mycorrhizal (AM) fungi is not known. GC–MS was used to investigate the fate of anthracene in the rhizosphere of ryegrass in a spiked soil with or without inoculation with AM fungi. GC–MS allowed us to identify and quantify an oxidation product of anthracene (anthraquinone) in soil. Oxidation of anthracene to anthraquinone was greater in planted than non-planted pots. The concentration of anthraquinone was lower in the mycorrhizal than non-mycorrhizal planted soil.

The ability of arbuscular mycorrhizal (AM) fungi from a metal-tolerant plant (Viola calaminaria, violet) to colonise and reduce metal uptake by a non-tolerant plant (Trifolium subterraneum, subterranean clover) in comparison to a metal-tolerant AM fungus isolated from a non-tolerant plant was studied. AM spores from the violet rhizosphere and from violet roots were characterised by polymerase chain reaction (PCR) amplification of the SSU rDNA, and sequencing. Subterranean clover was grown in pots containing a soil supplemented with Cd and Zn salts and inoculated either with a mixture of spores extracted from the violet rhizosphere or with spores of a Cd-tolerant Glomus mosseae P2 (BEG 69), or non-inoculated. The diversity of fungi, including AM fungi, colonising clover roots was assessed and analysed using terminal-restriction fragment length polymorphism. At least four different Glomus species were found in the violet rhizosphere. After 8 weeks in a growth chamber, colonisation of clover roots with spores from the violet rhizosphere increased Cd and Zn concentrations in clover roots without significantly affecting the concentrations of metals in the shoot and plant growth. G. mosseae P2 reduced plant growth and slightly increased the Cd concentration. Only one AM fungus (Glomus b) from the violet rhizosphere colonised clover roots, but other fungi were present. AM fungi from heavy metal-contaminated soils and associated with metal-tolerant plants may be effective in accumulating heavy metals in roots in a non-toxic form.

Plants may contribute to the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soils. Different mechanisms have been proposed, such as an increase in microbial numbers, but are not clearly elucidated. This study investigates the dissipation of a mixture of eight PAHs, ranging from 3 to 6 rings, in the rhizosphere of ryegrass (Lolium perenne L.). Two pot experiments were conducted with or without plants using soil spiked with 1 g kg−1 of PAHs in a growth chamber. The first experiment was carried out shortly after spiking and the second after 6 months of ageing. At the end of both experiments, the extractable concentrations of all PAHs were lower in rhizospheric than non-rhizospheric soil. PAH dissipation was lower after soil ageing than before, but was still significantly higher in the rhizospheric soil, even for three of the high molecular weight PAHs. Total culturable microflora were higher in the rhizospheric than non-rhizospheric soil, but was at the same level in spiked and non-spiked soil. The number of PAHs degraders, estimated by a modified MPN procedure, was not significantly different in the freshly spiked rhizospheric and non-rhizospheric soils, but was significantly higher in the rhizosphere of the aged spiked soil.

DAF (2,7-diaminofluorene)-peroxidases were the principal oxidative system in roots and rhizosphere of lucerne. The expression of these enzymes was stimulated by mycorrhiza as well as by anthracene (500 mg kg−1 added to the growth medium). Inversely, infection by mycorrhizal fungi repressed laccase activities. Electrophoretic analysis of the DAF-peroxidases revealed similar isoenzyme patterns for all treatments but in presence of anthracene some of the isoenzymes had more intensive bands.

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.

Experiments with excised mycelium of several Glomus spp. with different histories of exposure to heavy metals were carried out to measure their capacities to bind Cd and Zn. Cd sorption was followed for up to 6 h of incubation to determine its time course relationships. Controls treated with a metabolic inhibitor were included to evaluate whether sorption was due to active uptake or passive adsorption. The effect of ion competition (effects of Ca or Zn on Cd sorption) and general measurements of cation exchange capacity (CEC) of roots and hyphae were also performed. The results showed that AM mycelium has a high metal sorption capacity relative to other microorganisms, and a CEC comparable to other fungi. Metal sorption was rapid (

Arbuscular mycorrhizal (AM) fungi are known to have a beneficial effect on host plants growing in metal polluted soils. However, the potential contribution of AM fungi to cadmium (Cd) accumulation in plants is still uncertain and is rarely quantified in the context of phytoremediation. In a pot experiment, we quantified the partitioning of Cd in mycorrhizal (M) or non-mycorrhizal (NM) Medicago truncatula in four soils as a function of Cd availability. AM inoculation with Glomus intraradices increased plant growth and metal accumulation in above-ground biomass, even though Cd concentrations in M plants were lower than in NM plants. AM inoculation also increased the quantity of available Cd and reduced Cd in leachates. Results were consistent and significant in the four tested soils and were not related to Cd availability. Such AM fungi could be a good tool for phytoextraction.

High concentrations of heavy metals in soil have an adverse effect on micro-organisms and microbial processes. Among soil microorganisms, mycorrhizal fungi are the only ones providing a direct link between soil and roots, and can therefore be of great importance in heavy metal availability and toxicity to plants. This review discusses various aspects of the interactions between heavy metals and mycorrhizal fungi, including the effects of heavy metals on the occurrence of mycorrhizal fungi, heavy metal tolerance in these micro-organisms, and their effect on metal uptake and transfer to plants. Mechanisms involved in metal tolerance, uptake and accumulation by mycorrhizal hyphae and by endo- or ectomycorrhizae are covered. The possible use of mycorrhizal fungi as bioremediation agents in polluted soils or as bioindicators of pollution is also discussed.

SUMMARY Subterranean clover (Trifolium subterraneum L.) in symbiosis with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe was grown in compartmented pots. Side compartments were filled with soil where recently added (1 wk) radio-labelled cadmium (Cd) at three levels of non-labelled Cd could be accessed by either roots or arbuscular mycorrhizal hyphae. All treatments were replicated with non-mycorrhizal plants. After a growth period of 52 d roots and shoots were analysed for l09Cd, and lengths of roots and hyphae in respective labelled compartments determined. Cadmium uptake by roots was not significantly influenced by the mycorrhizal status of the plant. Uptake of Cd from hyphal compartments was higher in mycorrhizal than in non-mycorrhizal plants, corresponding to 96, 127 and 131 % of that in non-mycorrhizal plants when 1, 10 and 100 mg Cd kg-i was added, respectively. A large proportion of the increased Cd content of mycorrhizal plants was sequestered in the roots. It is concluded that extraradical hyphae of AM fungi can transport Cd from soil to plants, but that transfer from fungus to plant is restricted due to fungal immobilization. No reduction of hyphal growth into soil with up to 20 mg extractable Cd kg-' was observed.

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.

The bioavailability of heavy metals (Cd, Zn, Pb, Cu) and the abundance of arbuscular mycorrhiza (AM) were studied in two agricultural fields close to a Pb-Zn smelter and three fields outside the pollution zone all cultivated with maize (Zea mays L.). Metal extractability with ethylenediaminetetraacetic acid (EDTA)-NH4OAc and Ca(NO3)2, plant metal uptake, and mycorrhizal parameters (spore number, root colonization) were assessed at two growth stages (six-leaf and maturity). Despite regular liming, the availability of Cd, Zn, and Pb was markedly higher in the two metal-polluted fields than in the three uncontaminated fields. However, the AM abundance was not correlated with metal availability. Root colonization and spore numbers in the metal polluted fields were relatively high, though at plant maturity the former was significantly lower than in one of the uncontaminated fields. The very low AM abundance in the two other unpolluted fields was related to other factors, particular soil and plant P status and soil pH. AM root colonization did not substantially prevent plant metal accumulation, since the metal concentrations in maize grown on the polluted fields strongly exceeded normal values, and for Cd and Pb reached the limits of toxicity for animal feed.

International audience; Very little is known about the influence of bacterial-fungal ecological interactions on polycyclic aromatic hydrocarbon (PAH) dissipation in soils. Fusarium solani MM1 and Arthrobacter oxydans MsHM11 can dissipate PAHs in vitro. We investigated their interactions and their effect on the dissipation of three PAHs-phenanthrene (PHE), pyrene (PYR) and dibenz(a,h)anthracene (DBA)-in planted microcosms, in sterile sand or non-sterile soil. In sterile sand microcosms planted with alfalfa, the two microbes survived and grew, without any significant effect of co-inoculation. Co-inoculation led to the dissipation of 46 % of PHE after 21 days. In soil microcosms, whether planted with alfalfa or not, both strains persisted throughout the 46 days of the experiment, without any effect of co-inoculation or of alfalfa, as assessed by real-time PCR targeting taxon-level indicators, i.e. Actinobacteria 16S rDNA and the intergenic transcribed spacer specific to the genus Fusarium. The microbial community was analyzed by temporal temperature gradient electrophoresis and real-time PCR targeting bacterial and fungal rDNA and PAH-ring hydroxylating dioxygenase genes. These communities were modified by PAH pollution, which selected PAH-degrading bacteria, by the presence of alfalfa and, concerning the bacterial community, by inoculation. PHE and PYR concentrations significantly decreased (91 and 46 %, respectively) whatever the treatment, but DBA concentration significantly decreased (30 %) in planted and co-inoculated microcosms only.

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.

Spores of arbuscular mycorrhizal (AM) fungi were isolated from two heavy-metal polluted soils in France via trap culture with leek (Allium porrum L.). Preliminary identification showed that the predominant spore type of both cultures (P2 and Cd40) belongs to the Glomus mosseae group. Their sensitivity to cadmium was compared to a laboratory reference strain (G. mosseae) by in vitro germination tests with cadmium nitrate solutions at a range of concentrations (0 to 100 mg L−1) as well as extracts from a metal-polluted and unpolluted soils. Both cultures of AM fungi from heavy-metal polluted soils were more tolerant to cadmium than the G. mosseae reference strain. The graphically estimated EC50 was 0.8 mg L−1 Cd (concentration added to the test device) for G. mosseae and 7 mg L−1 for P2 culture, corresponding to effective Cd concentrations of approximately 50–70 μg L−1 and 200–500 μg L−1, respectively. The extract of the metal-polluted soil P2 decreased germination of spores from the reference G. mosseae but not from P2 culture. However, the extracts of two unpolluted soils with different physico-chemical characteristics did not affect G. mosseae, whereas germination of P2 spores was markedly decreased in the presence of one of the extracts. These results indicate a potential adaptation of AM fungi to elevated metal concentrations in soil. The tested spores may be considered as metal-tolerant ecotypes. Spore germination results in presence of soil extracts show the difficulty of assessing the ecotoxic effect of metals on AM fungi without considering other soil factors that may interfere in spore germination and hyphal extension.

The polycyclic aromatic hydrocarbon (PAH) contamination, bacterial community, and PAH-degrading bacteria were monitored in aged PAH-contaminated soil (Neuves-Maisons [NM] soil; with a mean of 1,915 mg of 16 PAHs·kg −1 of soil dry weight) and in the same soil previously treated by thermal desorption (TD soil; with a mean of 106 mg of 16 PAHs·kg −1 of soil dry weight). This study was conducted in situ for 2 years using experimental plots of the two soils. NM soil was colonized by spontaneous vegetation (NM-SV), planted with Medicago sativa (NM-Ms), or left as bare soil (NM-BS), and the TD soil was planted with Medicago sativa (TD-Ms). The bacterial community density, structure, and diversity were estimated by real-time PCR quantification of the 16S rRNA gene copy number, temporal thermal gradient gel electrophoresis fingerprinting, and band sequencing, respectively. The density of the bacterial community increased the first year during stabilization of the system and stayed constant in the NM soil, while it continued to increase in the TD soil during the second year. The bacterial community structure diverged among all the plot types after 2 years on site. In the NM-BS plots, the bacterial community was represented mainly by Betaproteobacteria and G ammaproteobacteria . The presence of vegetation (NM-SV and NM-Ms) in the NM soil favored the development of a wider range of bacterial phyla ( Alphaproteobacteria , Betaproteobacteria , G ammaproteobacteria , Verrucomicrobia , Actinobacteria , Firmicutes , and Chlorof l exi ) that, for the most part, were not closely related to known bacterial representatives. Moreover, under the influence of the same plant, the bacterial community that developed in the TD-Ms was represented by different bacterial species ( Alphaproteobacteria , Betaproteobacteria , Gammaproteobacteria , and Actinobacteria ) than that in the NM-Ms. During the 2 years of monitoring, the PAH concentration did not evolve significantly. The abundance of gram-negative (GN) and gram-positive (GP) PAH-degrading bacteria was estimated by real-time PCR quantification of specific functional genes encoding the α subunit of PAH-ring hydroxylating dioxygenase (PAH-RHD α ). The percentage of the PAH-RHD α GN bacterial genes relative to 16S rRNA gene density decreased with time in all the plots. The GP PAH-RHD α bacterial gene proportion decreased in the NM-BS plots but stayed constant or increased under vegetation influence (NM-SV, NM-Ms, and TD-Ms).

This review summarizes current knowledge on the contribution of mycorrhizal fungi to radiocesium immobilization and plant accumulation. These root symbionts develop extended hyphae in soils and readily contribute to the soil-to-plant transfer of some nutrients. Available data show that ecto-mycorrhizal (ECM) fungi can accumulate high concentration of radiocesium in their extraradical phase while radiocesium uptake and accumulation by arbuscular mycorrhizal (AM) fungi is limited. Yet, both ECM and AM fungi can transport radiocesium to their host plants, but this transport is low. In addition, mycorrhizal fungi could thus either store radiocesium in their intraradical phase or limit its root-to-shoot translocation. The review discusses the impact of soil characteristics, and fungal and plant transporters on radiocesium uptake and accumulation in plants, as well as the potential role of mycorrhizal fungi in phytoremediation strategies.

Improperly disposed of dredged sediments contaminated with metals may induce long-term leaching and an increase of metal concentrations in ground waters and vegetal cover plants. The objective of the study was to quantify the sediment-to-plant transfer of Cu, Pb, Hg and Zn with a particular focus on the pathway of Hg and to determine whether the establishment of vegetal cover modifies the metal availability. A pot experiment with rape (Brassica napus), cabbage (Brassica oleraccea) and red fescue (Festuca rubra) was set up using a sediment first spiked with the radioisotope 203Hg. Zinc concentrations (197-543 mg kg(-1) DM) in leaves were higher than Cu concentration (197-543 mg kg(-1) DM), Pb concentration (2.3-2.6 mg kg(-1) DM) and Hg concentration (0.9-1.7 mg kg(-1) DM). Leaves-to-sediment ratios decreased as follows: Zn > Cu > Hg > Pb. According to Ti measurements, metal contamination by dry deposition was less than 1%. Mercury concentration in plant leaves was higher than European and French thresholds. Foliar absorption of volatile Hg was a major pathway for Hg contamination with a root absorption of Hg higher in rape than in cabbage and red fescue. Growth of each species increased Cu solubility. Zinc solubility was increased only in the presence of rape. The highest increase of Cu solubility was observed for red fescue whereas this species largely decreased Zn solubility. Dissolved organic carbon (DOC) measurements suggested that Cu solubilisation could result from organic matter or release of natural plant exudates. Dissolved inorganic carbon (DIC) measures suggested that the high Zn solubility in the presence of rape could originate from a generation of acidity in rape rhizosphere and a subsequent dissolution of calcium carbonates. Consequently, emission of volatile Hg from contaminated dredged sediments and also the potential increase of metal solubility by a vegetal cover of grass when used in phytostabilisation must be taken into account by decision makers.

The diffuse pollution by fission and activation products following nuclear accidents and weapons testing is of major public concern. Among the nuclides that pose a serious risk if they enter the human food chain are the cesium isotopes 137 Cs and 134 Cs (with half-lives of 30 and 2 years, respectively). The biogeochemical cycling of these isotopes in forest ecosystems is strongly affected by their preferential absorption in a range of ectomycorrhiza-forming basidiomycetes. An even more widely distributed group of symbiotic fungi are the arbuscular mycorrhizal fungi, which colonize most herbaceous plants, including many agricultural crops. These fungi are known to be more efficient than ectomycorrhizas in transporting mineral elements from soil to plants. Their role in the biogeochemical cycling of Cs is poorly known, in spite of the consequences that fungal Cs transport may have for transfer of Cs into the human food chain. This report presents the first data on transport of Cs by these fungi by use of radiotracers and compartmented growth systems where uptake by roots and mycorrhizal hyphae is distinguished. Independent experiments in three laboratories that used different combinations of fungi and host plants all demonstrated that these fungi do not contribute significantly to plant uptake of Cs. The implications of these findings for the bioavailability of radiocesium in different terrestrial ecosystems are discussed.

Plant secondary metabolites, such as saponins, have a considerable impact in agriculture because of their allelopathic effects. They also affect the growth of soil microorganisms, especially fungi. We investigated the influence of saponins on rhizosphere bacteria in vitro and in soil conditions. The effects of gypsophila saponins on the growth kinetics of rhizosphere bacteria were studied by monitoring the absorbance of the cultures in microtiter plates. Gypsophila saponins (1%) increased the lag phase of bacterial growth. The impact of gypsophila saponins on subterranean clover rhizosphere was also investigated in a pot experiment. The addition of gypsophila saponins did not modify clover biomass but significantly increased (twofold with 1% saponins) the weight of adhering soil. The number of culturable heterotrophic bacteria of the clover rhizosphere was not affected by the addition of gypsophila saponins. Nevertheless, the phenotypical characterization of the dominant Gram-negative strains of the clover rhizosphere, using the Biolog©system, showed qualitative and quantitative differences induced by 1% saponins. With the addition of saponins, the populations of Chryseomonas spp. and Acinetobacter spp., the two dominant culturable genera of control clover, were no longer detectable or were significantly decreased, while that of Aquaspirillum dispar increased and Aquaspirillum spp. became the major genus. Aquaspirillum dispar and Aquaspirillum spp. were also the dominant rhizosphere bacteria of Gypsophila paniculata, which greatly accumulates these saponins in its roots. These results suggest that saponins may control rhizosphere bacteria in soil through rhizodeposition mechanisms.Key words: bacteria, Gypsophila paniculata, rhizosphere, saponins, Trifolium subterraneum.

Fungi play crucially important roles in the biosphere, mediating many ecological processes. Despite this, the fungal diversity in natural habitats is poorly known. Using newly designed fungal-specific polymerase chain reaction (PCR) primers ([1][1]), we examined total fungal diversity in a

The genetic diversity of spores of two indigenous species of Glomus isolated from three soils of a longterm field experiment amended by different quantities of sewage sludges has been evaluated. Three populations of spores of Glomus claroideum (W2537) and three populations of spores of Glomus DAOM 225952 (W2538) were analysed using a microsatellite primer and aliquots of genomic DNA were obtained from single spores (Inter Simple Sequence, Repeat (ISSR) fingerprints). 39 polymorphic bands were found for G. claroideum, and 43 in Glomus DAOM 225952. The intraspecific diversity was high, ranging from 22 to 33 different electrophoretic types for G. claroideum. and 15-27 for Glomus DAOM 225952 depending on the population. Resampling experiments showed that the number of polymorphic bands was sufficient to score all multilocus profiles in the populations and to describe the clonality structure within populations. On average, one multilocus profile was represented by about four spores whatever the Population and the species. Partitioning of the within-species phenotypic variance showed that more than 92% of the variation was found, within populations., while the among-population variance component accounted for less than 8%, even though it was statistically different from 0. This result is confirmed by the fact that only few multilocus profiles were shared by two populations of G. claroideum. and none by populations of Glomus DAOM 225952. In addition to the high level of diversity observed within populations, linkage disequilibria analyses and association indices calculated across loci indicates that reproduction cannot be solely clonal. Recombination or recombination-like events are likely to occur in these arbuscular mycorrhizal fungi. An 'epidemic' population structure was found for both fungal species in the soil that had received high amounts of sewage sludge.

Phytoremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs) has so far neglected the possible role of the ubiquitous symbiotic associations between plant roots and fungi known as arbuscular mycorrhizas. A time course laboratory experiment with clover and ryegrass grown on spiked [500 + 500 + 50 mg kg-1 of anthracene, chrysene and dibenz(a,h)anthracene] soil demonstrated for the first time that dissipation of condensed PAHs may be enhanced in the presence of arbuscular mycorrhiza [66 and 42% reductions in chrysene and dibenz(a,h)anthracene, respectively, versus 56 and 20% reductions in nonmycorrhizal controls]. Addition of a surfactant accelerated initial PAH dissipation but did not attain final PAH concentrations below those obtained with nonmycorrhizal plants. Toxicity tests (earthworm survival and bioluminescence inhibition in Vibrio fischeri) indicated that mycorrhiza reduced the toxicity of PAHs and/or their metabolites and counteracted a temporally enhanced toxicity mediated by surfactant addition. Phospholipid fatty acid profiles demonstrated that the imposed treatments altered the microbial community structure and indicated that the mycorrhiza-associated microflora was responsible for the observed reductions in PAH concentrations in the presence of mycorrhiza.

Among soil microorganisms, arbuscular-mycorrhizal (AM) fungi are ubiquitous in agriculturous soils and some natural ecosystems. They are known to promote plant growth and plant uptake of nutrients. In contaminated soils they can play an important role since they provide a direct link between soil and plant roots. Little attention has been given to these fungi in bioremediation studies or as bioassay for soil and especially rhizospheric soil contamination.