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ABSTRACTHigh-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity.

Marginal lands have been proposed to produce non-food crop biomass for energy or green materials. For this purpose, the selection, implementation, and growth optimization of plant species on such lands are key elements to investigate to achieve relevant plant yields. Stinging nettle (Urtica dioica) is a herbaceous perennial that grows spontaneously on contaminated lands and was described as suitable to produce fibers for material applications. Two mercury-contaminated soils from industrial wastelands with different properties (grassland soil and sediment landfill) were used in this study to assess the potential growth of stinging nettle in a greenhouse mesocosm experiment. Two organic amendments were studied for their impact on nettle growth. The solid digestate from organic food wastes significantly doubled plant biomass whereas the compost from green wastes had a lower impact. The highest doses of organic amendments significantly increased the number of fibers, which doubled following digestate application, while reducing leaf Hg concentration. Both amendments significantly improved soil respiration and enzymatic activities linked to the microbial biomass in the soil from the sediment landfill by the end of the experiment. In the context of a phytomanagement scenario, solid digestate would be a preferred amendment resource to improve nettle production on industrial wastelands.

Mercury (Hg) pollution is a global issue due to the high toxicity and wide dispersion of Hg around the world. Whether due to anthropogenic activities or natural processes, Hg emissions are steadily increasing, with very high levels in some regions, directly threatening human and ecosystem health. However, bacteria and fungi have evolved and adapted in response to Hg-induced stress and have developed tolerance mechanisms, notably based on the mer operon system that is involved in Hg uptake and biovolatilization via Hg reduction reactions. Other processes, such as bioaccumulation or extracellular sequestration, are involved in Hg resistance, and the study of contaminated soils has allowed the isolation of a number of microorganisms capable of these mechanisms, with strong potential for the implementation of bioremediation approaches. In addition to playing an important role in determining the fate of Hg in the biogeochemical cycle, these microorganisms can indeed be applied to reduce Hg concentrations or at least stabilize Hg for the remediation of polluted soils. Moreover, thanks to the development of biotechnological tools, bioremediation based on Hg-tolerant microorganisms can be optimized. Finally, these microorganisms are relevant candidates for biomonitoring, for example, through the engineering of biosensors, because the detection of Hg is a major issue in preserving the health of living beings.

AimsAfforestation of trace-element contaminated soils, notably with fast growing trees, has been demonstrated to be an attractive option for bioremediation due to the lower costs and dispersion of contaminants than conventional cleanup methods. Mycorrhizal fungi form symbiotic associations with plants, contributing to their tolerance towards toxic elements and actively participating to the biorestoration processes. The aim of this study was to deepen our understanding on the effects of mycorrhizal inoculation on plant development and fungal community at two trace-element contaminated sites (Pierrelaye and Fresnes-sur-Escaut, France) planted with poplar (Populus trichocarpa x Populus maximowiczii).MethodsThe 2 sites were divided into 4 replicated field blocks with a final plant density of 2200 tree h-1. Half of the trees were inoculated with a commercial inoculum made of a mix of mycorrhizal species. The sites presented different physico-chemical characteristics (e.g., texture: sandy soil versus silty-loam soil and organic matter: 5.7% versus 3.4% for Pierrelaye and Fresnes-sur-Escaut, respectively) and various trace element contamination levels.ResultsAfter 7 years of plantation, inoculation showed a significant positive effect on poplar biomass production at the two sites. Fungal composition study demonstrated a predominance of the phylum Ascomycota at both sites, with a dominance of Geopora Arenicola and Mortierella elongata, and a higher proportion of ectomycorrhizal and endophytic fungi (with the highest values observed in Fresnes-sur-Escaut: 45% and 28% for ECM and endophytic fungi, respectively), well known for their capacity to have positive effects on plant development in stressful conditions. Furthermore, Pierrelaye site showed higher frequency (%) of mycorrhizal tips for ectomycorrhizal fungi (ECM) and higher intensity (%) of mycorrhizal root cortex colonization for arbuscular mycorrhizal fungi (AMF) than Fresnes-sur-Escaut site, which translates in a higher level of diversity.ConclusionsFinally, this study demonstrated that this biofertilization approach could be recommended as an appropriate phytomanagement strategy, due to its capacity to significantly improve poplar productivity without any perturbations in soil mycobiomes.

The rapidly expanding utilization of lanthanides (Ln) for the development of new technologies, green energies, and agriculture has raised concerns regarding their impacts on the environment and human health. The absence of characterization of the underlying cellular and molecular mechanisms regarding their toxicity is a caveat in the apprehension of their environmental impacts. We performed genomic phenotyping and molecular physiology analyses of Saccharomyces cerevisiae mutants exposed to La and Yb to uncover genes and pathways affecting Ln resistance and toxicity. Ln responses strongly differed from well-known transition metal and from common responses mediated by oxidative compounds. Shared response pathways to La and Yb exposure were associated to lipid metabolism, ion homeostasis, vesicular trafficking, and endocytosis, which represents a putative way of entry for Ln. Cell wall organization and related signaling pathways allowed for the discrimination of light and heavy Ln. Mutants in cell wall integrity-related proteins (e.g., Kre1p, Kre6p) or in the activation of secretory pathway and cell wall proteins (e.g., Kex2p, Kex1p) were resistant to Yb but sensitive to La. Exposure of WT yeast to the serine protease inhibitor tosyl phenylalanyl chloromethyl ketone mimicked the phenotype of kex2∆ under Ln, strengthening these results. Our data also suggest that the relative proportions of chitin and phosphomannan could modulate the proportion of functional groups (phosphates and carboxylates) to which La and Yb could differentially bind. Moreover, we showed that kex2∆, kex1∆, kre1∆, and kre6∆ strains were all sensitive to light Ln (La to Eu), while being increasingly resistant to heavier Ln. Finally, shotgun proteomic analyses identified modulated proteins in kex2∆ exposed to Ln, among which several plasmalemma ion transporters that were less abundant and that could play a role in Yb uptake. By combining these different approaches, we unraveled that cell wall components not only act in Ln adsorption but are also active signal effectors allowing cells to differentiate light and heavy Ln. This work paves the way for future investigations to the better understanding of Ln toxicity in higher eukaryotes.

The increasing production of wastes that are landfilled might contribute to sources of potentially toxic elements; this is the case of residual red gypsum tailings, a by-product of titanium dioxide extraction. Revegetation of such a site is essential, and Mn phytoextraction may render the operations economically profitable. This study aimed to apply phytomanagement techniques for increasing the plant development, tailings revegetation and an optimal Mn phytoextraction using silver birch, the most abundant plant species on this site. To enhance the nutrient availability from the tailings, amendments that reduce the pH, i.e., pine bark chips, Miscanthus straw, white peat, and ericaceous compost, were mixed with residual red gypsum and birches were allowed to grow for 3 months. The pine bark chips and ericaceous compost led to a maximum decrease in pH, allowing the accumulation of up to 1400 mg Mn kg–1 dry matter in the leaves silver birch leaves. However, some nutrient competition was found in the pine bark treatment, which halved biomass production as compared to control. Further amendment addition may be needed to take advantage of the pine bark capabilities as a soil conditioner and Mn solubilizing treatment in residual red gypsum.

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.

The successful restoration of well-engineered tailings storage facilities is needed to avoid mine tailings problems. This study characterized the bacterial communities from vegetated and non-vegetated soils from a red gypsum landfill resulting from the industrial extraction of titanium. A set of 275 bacteria was isolated from vegetated soil and non-vegetated soil areas and taxonomically characterized using BOX-PCR. The study also evaluated the ability of a subset of 88 isolated bacteria on their ability to produce plant growth promoting (PGP) traits [indoleacetic acid (IAA) production, phosphate solubilization, and siderophore production] and their tolerance to potentially toxic elements (PTEs). Twenty strains were chosen for further analysis to produce inoculum for birch-challenging experiments. Principal component analysis (PCA) showed that the set of pedological parameters (pH, granulometry, carbon, organic matter, and Mg content) alone explained approximately 40% of the differences between the two soils. The highest density of total culturable bacteria was found in the vegetated soil, and it was much higher than that in the non-vegetated soil. The Actinobacteria phyla dominated the culturable soil community (70% in vegetated soil and 95% in non-vegetated soil), while the phyla Firmicutes (including the genus Bacillus) and Bacteroides (including the genera Pedobacter and Olivibacter) were found only in the vegetated soil fraction. Additional genera (Rhizobium, Variovorax, and Ensifer) were found solely in the vegetated soil. The vegetated soil bacteria harbored the most beneficial PGP bacteria with 12% of the isolates showing three or more PGP traits. The strains with higher metal tolerances in our study were Phyllobacterium sp. WR140 (RO1.15), Phyllobacterium sp. WR140 (R01.34), and Streptomyces sp. (R04.15), all isolated from the vegetated soil. Among the isolates tested in challenging experiments, Phyllobacterium (R01.34) and Streptomyces sp. (R05.33) have the greatest potential to act as PGP rhizobacteria and therefore to be used in the biological restoration of tailings dumps.

Mycorrhizas of Pinus sylvestris, collected from zinc wastes in Poland and France were investigated using transmission electron microscope (TEM) and scanning electron microscope (SEM) equipped with energy dispersion spectroscopy (EDS) and electron energy loss spectroscopy (EELS). At both sites, mycorrhizas of Hebeloma were the most frequent, however, they were often characterised by a sparse or only locally developed fungal mantle. Mycorrhizas formed by suilloid fungi were much less frequent, and usually produced a clearly defined fungal mantle characterised by abundant formation of pigments and crystals covering the hyphae of the outer mantle. These two groups of mycorrhizas differed in their heavy metal binding properties. A biofiltering effect of Pb and Zn by the fungal mantle was observed only in the case of suilloid mycorrhizas, which represented up to 10% of the total number of mycorrhizas. No statistical differences between the mantle, the cortical cell walls and the vascular tissue were demonstrated in mycorrhizas formed by other fungi dominating on industrial wastes. In the case of Hebeloma and Inocybe, however, elements such as Cu and Cd were present in higher amounts in the extra-matrical mycelium, whereas no or only low amounts of these elements were detected within fungal mantles, mainly in mycorrhizas from the French waste. Analysis of the root systems has shown relatively high percentage of nonmycorrhizal short roots, suggesting the inhibition of mycorrhiza formation or a decreased number of mycorrhizal propagules. The role of dead roots and mycorrhizas in biosorption and immobilization of heavy metals was discussed.

Soil nitrogen can alter storage and remobilization of carbon and nitrogen in forest trees and affect growth responses to elevated carbon dioxide concentration ([CO2]). We investigated these effects in oak saplings (Quercus robur L.) exposed for two years to ambient or twice ambient [CO2] in combination with low- (LN, 0.6 mmol N l−1) or high-nitrogen (HN, 6.1 mmol N l−1) fertilization. Autumn N retranslocation efficiency from senescing leaves was less in HN saplings than in LN saplings, but about 15% of sapling N was lost to the litter. During the dormant season, nonstructural carbohydrates made up 20 to 30% of the dry mass of perennial organs. Starch was stored mainly in large roots where it represented 35–46% of dry mass. Accumulation of starch increased in large roots in response to LN but was unaffected by elevated [CO2]. The HN treatment resulted in high concentrations of N-soluble compounds, and this effect was reduced by elevated [CO2], which decreased soluble protein N (−17%) and amino acid N (−37%) concentrations in the HN saplings. Carbon and N reserves were labeled with 13C and 15N, respectively, at the end of the first year. In the second year, about 20% of labeled C and 50% of labeled N was remobilized for spring growth in all treatments. At the end of leaf expansion, 50–60% of C in HN saplings originated from assimilation versus only 10–20% in LN saplings. In HN saplings only, N uptake occurred, and some newly assimilated N was allocated to new shoots. Through effects on the C and N content of perennial organs, elevated [CO2] and HN increased remobilization capacity, thereby supporting multiple shoot flushes, which increased leaf area and subsequent C acquisition in a positive feedback loop. [ABSTRACT FROM AUTHOR]