Transfer of La, Ce, Sm and Yb to alfalfa and ryegrass from spiked soil and the role of Funneliformis mosseae.

Rare earth elements (REEs) are widely used in high-tech industries, and REE waste emissions have become a concern for ecosystems, food quality and human beings. Arbuscular mycorrhizal fungi (AMF) have repeatedly been reported to alleviate plant stress in metal-contaminated soils. To date, little information is available concerning the role of AMF in REE-contaminated soils. We recently showed that there was no transfer of Sm to alfalfa by Funneliformis mosseae, but only a single REE was examined, while light and heavy REEs are present in contaminated soils. To understand the role of AMF on the transfer of REEs to plants, we carried out an experiment using alfalfa (Medicago sativa) and ryegrass (Lolium perenne) in compartmented pots with separate bottom compartments that only were accessible by F. mosseae fungal hyphae. The bottom compartments contained a mixture of four REEs at equal concentrations (La, Ce, Sm and Yb). The concentration of REEs in plants was higher in roots than in shoots with higher REE soil-root than root-shoot transfer factors. Moreover, significantly higher light-REEs La and Ce were transferred to ryegrass shoots than Sm and the heavy-REE Yb, but this was not observed for alfalfa. Alfalfa dry weight was significantly increased by F. mosseae inoculation, but not ryegrass dry weight. For both plant species, there was significantly higher P uptake by the mycorrhizal plants than the nonmycorrhizal plants, but there was no significant transfer of La, Ce, Sm or Yb to alfalfa and ryegrass roots or shoots due to F. mosseae inoculation.

No significant transfer of the rare earth element samarium from spiked soil to alfalfa by Funneliformis mosseae.

Rare earth elements including samarium have been widely used in modern technologies in recent decades. Following over-exploitation and soil contamination, they can accumulate in plants and be toxic at high concentrations. Arbuscular mycorrhizae benefit plants in metal-contaminated soils by improving their survival and growth and alleviating metal toxicity, but little information is available about soil contaminated by rare earth elements. We performed two experiments using samarium to study the role of arbuscular mycorrhizal fungi on plant growth and samarium transfer to alfalfa in a samarium-spiked soil. A pot experiment was conducted in a soil spiked with two concentrations of samarium and a non-spiked control, inoculated or not with a metal-tolerant Funneliformis mosseae. A compartmented pot experiment was then performed with a separated compartment containing samarium-spiked sand only accessible by F. mosseae fungal hyphae to further study the transport of samarium from the soil to alfalfa. The biomass of alfalfa grown on samarium-spiked soil was reduced, while it was significantly higher following arbuscular mycorrhiza inoculation in the pot experiment, both in the control and samarium-spiked soil. Although mycorrhizal plants had a higher phosphorus content than non-mycorrhizal ones, there was no significant difference in samarium concentrations between mycorrhizal and non-mycorrhizal plants. The compartment experiment confirmed that there was no significant samarium transfer to the plant by F. mosseae. Other fungi and plants should be tested, and field experiments performed, but our results suggest that arbuscular mycorrhizal plants might be considered in phytorestoration of rare-earth-contaminated soils.

Soil Properties and Multi-Pollution Affect Taxonomic and Functional Bacterial Diversity in a Range of French Soils Displaying an Anthropisation Gradient.

The intensive industrial activities of the twentieth century have left behind highly contaminated wasteland soils. It is well known that soil parameters and the presence of pollutants shape microbial communities. But in such industrial waste sites, the soil multi-contamination with organic (polycyclic aromatic hydrocarbons, PAH) and metallic (Zn, Pb, Cd) pollutants and long-term exposure may induce a selection pressure on microbial communities that may modify soil functioning. The aim of our study was to evaluate the impact of long-term multi-contamination and soil characteristics on bacterial taxonomic and functional diversity as related to the carbon cycle. We worked on 10 soils from northeast of France distributed into three groups (low anthropised controls, slag heaps, and settling ponds) based on their physico-chemical properties (texture, C, N) and pollution level. We assessed bacterial taxonomic diversity by 16S rDNA Illumina sequencing, and functional diversity using Biolog® and MicroResp™ microtiter plate tools. Although taxonomic diversity at the phylum level was not different among the soil groups, many operational taxonomic units were influenced by metal or PAH pollution, and by soil texture and total nitrogen content. Functional diversity was not influenced by PAH contamination while metal pollution selected microbial communities with reduced metabolic functional diversity but more tolerant to zinc. Limited microbial utilisation of carbon substrates in metal-polluted soils was mainly due to the nitrogen content. Based on these two observations, we hypothesised that reduced microbial activity and lower carbon cycle-related functional diversity may have contributed to the accumulation of organic matter in the soils that exhibited the highest levels of metal pollution.

Co-inoculation of Lolium perenne with Funneliformis mosseae and the dark septate endophyte Cadophora sp. in a trace element-polluted soil.

The presence of dark septate endophytes (DSEs) or arbuscular mycorrhizal fungi (AMF) in plant roots and their effects on plant fitness have been extensively described. However, little is known about their interactions when they are simultaneously colonizing a plant root, especially in trace element (TE)-polluted soils. We therefore investigated the effects of Cadophora sp. and Funneliformis mosseae on ryegrass (Lolium perenne) growth and element uptake in a Cd/Zn/Pb-polluted soil. The experiment included four treatments, i.e., inoculation with Cadophora sp., inoculation with F. mosseae, co-inoculation with Cadophora sp. and F. mosseae, and no inoculation. Ryegrass biomass and shoot Na, P, K, and Mg concentrations significantly increased following AMF inoculation as compared to non-inoculated controls. Similarly, DSE inoculation increased shoot Na concentration, whereas dual inoculation significantly decreased shoot Cd concentration. Moreover, oxidative stress determined by ryegrass leaf malondialdehyde concentration was alleviated both in the AMF and dual inoculation treatments. We used quantitative PCR and microscope observations to quantify colonization rates. They demonstrated that DSEs had no effect on AMF colonization, while AMF colonization slightly decreased DSE frequency. We also monitored fluorescein diacetate (FDA) hydrolysis and alkaline phosphatase (AP) activity in the rhizosphere soils. FDA hydrolysis remained unchanged in the three inoculated treatments, but AMF colonization increased AP activity and P mobility in the soil whereas DSE colonization did not alter AP activity. In this experiment, we unveiled the interactions between two ecologically important fungal groups likely to occur in roots which involved a decrease of oxidative stress and Cd accumulation in shoots. These results open promising perspectives on the fungal-based phytomanagement of TE-contaminated sites by the production of uncontaminated and marketable plant biomass.

Rhizosphere effect is stronger than PAH concentration on shaping spatial bacterial assemblages along centimetre-scale depth gradients.

At centimetre scale, soil bacterial assemblages are shaped by both abiotic (edaphic characteristics and pollutants) and biotic parameters. In a rhizobox experiment carried out on planted industrial soil contaminated with polycyclic aromatic hydrocarbons (PAHs), we previously showed that pollution was distributed randomly with hot and cold spots. Therefore, in the present study, we investigated the effect of this patchy PAH distribution on the bacterial community assemblage and compared it with that of root depth gradients found in the rhizosphere of either alfalfa or ryegrass. Sequencing of 16S rRNA amplicons revealed a higher bacterial diversity in ryegrass rhizosphere and enrichment in specific taxa by the 2 plant species. Indeed, Bacteroidetes, Firmicutes, and Gammaproteobacteria were globally favored in alfalfa, whereas Acidimicrobiia, Chloroflexi, Alpha-, and Betaproteobacteria were globally favored in ryegrass rhizosphere. The presence of alfalfa created depth gradients of root biomass, carbohydrate, and pH, and actually shaped the bacterial assemblage, favoring Actinobacteria near the surface and Gemmatimonadetes and Proteobacteria at greater depths. Contrarily, the bacterial assemblage was homogeneous all along depths of the ryegrass root system. With both plant species, the PAH content and random distribution had no significant effect on bacterial assemblage. Globally, at centimeter scale, bacterial community assemblages were mostly shaped by soil physical and chemical depth gradients induced by root growth but not by patchy PAH content.

Differential growth promotion of poplar and birch inoculated with three dark septate endophytes in two trace element-contaminated soils.

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.

The Bacterial and Fungal Diversity of an Aged PAH- and Heavy Metal-Contaminated Soil is Affected by Plant Cover and Edaphic Parameters.

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).

Inoculation of PAH-degrading strains of Fusarium solani and Arthrobacter oxydans in rhizospheric sand and soil microcosms: microbial interactions and PAH dissipation.

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.

Root exudates modify bacterial diversity of phenanthrene degraders in PAH-polluted soil but not phenanthrene degradation rates.

To determine whether the diversity of phenanthrene-degrading bacteria in an aged polycyclic aromatic hydrocarbon (PAH) contaminated soil is affected by the addition of plant root exudates, DNA stable isotope probing (SIP) was used. Microcosms of soil with and without addition of ryegrass exudates and with ¹³C-labelled phenanthrene (PHE) were monitored over 12 days. PHE degradation was slightly delayed in the presence of added exudate after 4 days of incubation. After 12 days, 68% of added PHE disappeared both with and without exudate. Carbon balance using isotopic analyses indicated that a part of the ¹³C-PHE was not totally mineralized as ¹³CO2 but unidentified ¹³C-compounds (i.e. ¹³C-PHE or ¹³C-labelled metabolites) were trapped into the soil matrix. Temporal thermal gradient gel electrophoresis (TTGE) analyses of 16S rRNA genes were performed on recovered ¹³C-enriched DNA fractions. 16S rRNA gene banding showed the impact of root exudates on diversity of PHE-degrading bacteria. With PHE as a fresh sole carbon source, Pseudoxanthomonas sp. and Microbacterium sp. were the major PHE degraders, while in the presence of exudates, Pseudomonas sp. and Arthrobacter sp. were favoured. These two different PHE-degrading bacterial populations were also distinguished through detection of PAH-ring hydroxylating dioxygenase (PAH-RHD(α)) genes by real-time PCR. Root exudates favoured the development of a higher diversity of bacteria and increased the abundance of bacteria containing known PAH-RHD(α) genes.

Altered fungal communities in contaminated soils from French industrial brownfields.

Polycyclic aromatic hydrocarbons (PAHs) and metals are contaminants of industrial brownfield soils. Pollutants can have harmful effects on fungi, which are major actors of soil functioning. Our objective was to highlight fungal selection following long-term contamination of soils. Fungal diversity was assessed on 30 top-soil samples from ten sites gathered in three groups with different contamination levels and physico-chemical characteristics: 1) uncontaminated controls, 2) slag heaps displaying high PAH and moderate metal contaminations, and 3) settling ponds displaying high metal and intermediate PAH contaminations. Although fungal abundance and richness were similar among the soil groups, the diversity and evenness indices were lower for the slag heap group. Fungal diversity differed among soil groups at the phylum and OTU levels, and indicator species were identified. The relative abundance of Agaricomycetes, Saccharomycetes, Leotiomycetes and Chytridiomycota was higher in the control soils than in the two groups of contaminated soils. Cryptomycota LKM11 representatives were favoured in the slag heap and settling pond groups, and their relative abundance was correlated to the zinc and lead contamination levels. Dothideomycetes - positively linked to PAH contamination - and Eurotiomycetes were specific to the slag heap group. Pucciniomycetes and especially Gymnosporangium members were favoured in the settling pond soils.

Population structure and abundance of arsenite-oxidizing bacteria along an arsenic pollution gradient in waters of the upper isle River Basin, France.

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta- and Gammaproteobacteria, depending on arsenic (As) and E(h) levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters.

Bioavailability and transfer of elevated Sm concentration to alfalfa in spiked soils.

Rare earth elements (REEs) have been widely used in recent decades, and their exploitation has led to industrial REE emission and to contaminated soils especially in former mining areas. This raised people concerns on the accumulation and toxicity of REEs in soils and plants, and consequences on plant health. Although many studies dealt with REE in soils and plants, there is still a need to precise their toxicity, bioavailability and transfer to plants in contaminated sites in order to restore such ecosystems. We studied the bioavailability and transfer of a REE to Medicago sativa grown on two contaminated soils differing in their chemical characteristics. A pot experiment was set up in a growth chamber where two natural soils were spiked or not with samarium (Sm) as a model REE. Two chemical extractants were tested to estimate the bioavailability of Sm in the soil, its decrease with time and its transfer to the plants. Results showed that DTPA extractable Sm was well correlated with Sm uptake in alfalfa shoots. The experiment pointed out a significant ageing effect since DTPA extractable Sm significantly decreased within 2 weeks in the soils and was significantly lower in the less acidic soil than in the other. The uptake of Sm from soil to alfalfa shoots depended on the soil pH and on the spiking concentration. The soil to plant transfer factor was low (< 0.08), but a 30% reduction of alfalfa biomass was observed when the soils were spiked with 100 to 200 mg kg-1 of Sm.

Unravelling the Role of Melanin in Cd and Zn Tolerance and Accumulation of Three Dark Septate Endophytic Species.

Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera Cadophora sp., Leptodontidium sp. and Phialophora mustea, were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of Leptodontidium sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of Leptodontidium albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.

Influence of vegetation on the in situ bacterial community and polycyclic aromatic hydrocarbon (PAH) degraders in aged PAH-contaminated or thermal-desorption-treated soil.

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 Gammaproteobacteria. 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, Gammaproteobacteria, Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi) 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 alpha subunit of PAH-ring hydroxylating dioxygenase (PAH-RHD(alpha)). The percentage of the PAH-RHD(alpha) GN bacterial genes relative to 16S rRNA gene density decreased with time in all the plots. The GP PAH-RHD(alpha) bacterial gene proportion decreased in the NM-BS plots but stayed constant or increased under vegetation influence (NM-SV, NM-Ms, and TD-Ms).

DNA stable isotope probing reveals contrasted activity and phenanthrene-degrading bacteria identity in a gradient of anthropized soils.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous soil organic pollutants. Although PAH-degrading bacteria are present in almost all soils, their selection and enrichment have been shown in historically high PAH contaminated soils. We can wonder if the effectiveness of PAH biodegradation and the PAH-degrading bacterial diversity differ among soils. The stable isotope probing (SIP) technique with 13C-phenanthrene (PHE) as a model PAH was used to: (i) compare for the first time a range of 10 soils with various PAH contamination levels, (ii) determine their PHE-degradation efficiency and (iii) identify the active PHE-degraders using 16S rRNA gene amplicon sequencing from 13C-labeled DNA. Surprisingly, the PHE degradation rate was not directly correlated to the initial level of total PAHs and phenanthrene in the soils, but was mostly explained by the initial abundance and richness of soil bacterial communities. A large diversity of PAH-degrading bacteria was identified for seven of the soils, with differences among soils. In the soils where the PHE degradation activities were the higher, Mycobacterium species were always the dominant active PHE degraders. A positive correlation between PHE-degradation level and the diversity of active PHE-degraders (Shannon index) supported the hypothesis that cooperation between strains led to a more efficient PAH degradation.

Interactions between dark septate endophytes, ectomycorrhizal fungi and root pathogens in vitro.

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.

Real-Time PCR quantification of PAH-ring hydroxylating dioxygenase (PAH-RHDalpha) 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.

Assessment of derelict soil quality: Abiotic, biotic and functional approaches.

The intensification and subsequent closing down of industrial activities during the last century has left behind large surfaces of derelict lands. Derelict soils have low fertility, can be contaminated, and many of them remain unused. However, with the increasing demand of soil surfaces, they might be considered as a resource, for example for non-food biomass production. The study of their physico-chemical properties and of their biodiversity and biological activity may provide indications for their potential re-use. The objective of our study was to investigate the quality of six derelict soils, considering abiotic, biotic, and functional parameters. We studied (i) the soil bacteria, fungi, meso- and macro-fauna and plant communities of six different derelict soils (two from coking plants, one from a settling pond, two constructed ones made from different substrates and remediated soil, and an inert waste storage one), and (ii) their decomposition function based on the decomposer trophic network, enzyme activities, mineralization activity, and organic pollutant degradation. Biodiversity levels in these soils were high, but all biotic parameters, except the mycorrhizal colonization level, discriminated them. Multivariate analysis showed that biotic parameters co-varied more with fertility proxies than with soil contamination parameters. Similarly, functional parameters significantly co-varied with abiotic parameters. Among functional parameters, macro-decomposer proportion, enzyme activity, average mineralization capacity, and microbial polycyclic aromatic hydrocarbon degraders were useful to discriminate the soils. We assessed their quality by combining abiotic, biotic, and functional parameters: the compost-amended constructed soil displayed the highest quality, while the settling pond soil and the contaminated constructed soil displayed the lowest. Although differences among the soils were highlighted, this study shows that derelict soils may provide a biodiversity ecosystem service and are functional for decomposition.

High PAH degradation and activity of degrading bacteria during alfalfa growth where a contrasted active community developed in comparison to unplanted soil.

PAH biodegradation in plant rhizosphere has been investigated in many studies, but the timescale of degradation and degrading bacteria activity was rarely considered. We explored the impact of plants on the temporal variability of PAH degradation, microbial abundance, activity, and bacterial community structure in a rhizotron experiment. A historically contaminated soil was spiked with PAHs, planted or not with alfalfa, over 22 days with sampling once a week. In both conditions, most of the spiked PAHs were dissipated during the first week, conducting to polar polycyclic aromatic compound production and to decreased richness and diversity of bacterial communities. We showed a rapid impact of the rhizosphere on PAH degradation via the increased activity of PAH-degrading bacteria. After 12 days, PAH degradation was significantly higher in the planted (100% degradation) than in unplanted (70%) soil. Gram-negative (Proteobacteria) PAH-dioxygenase genes and transcripts were higher in planted than unplanted soil and were correlated to the spiked PAH degradation. Conversely, Gram-positive (Actinobacteria) PAH-dioxygenase gene transcription was constant over time in both conditions. At 12 days, plant growth favored the activity of many Gammaproteobacteria (Pseudomonadaceae, Stenotrophomonas, and Acinetobacter) while in unplanted soil Alphaproteobacteria (Sphingomonadaceae, Sphingobium, and Magnetospirillum) and Actinobacteria (Iamia, Geodermatophilaceae, and Solirubrobacterales) were more active.

Melanization and ageing are not drawbacks for successful agro-transformation of dark septate endophytes.

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 %. 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 15-d-old mycelium (44.5, 6.9, and 1.1 %, respectively) as compared to 2-d-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 g-1 DW. Tricyclazole negatively modulated melanin content, while copper positively modulated it. However there was no correlation between hyphal melanin content and ATMT efficiency.