Vanessa Álvarez-López

Cyril Zappelini

Alexis Durand

Michel Chalot

Pioneer tress of Betula pendula at a red gypsum landfill harbour specific structure and composition of root-associated microbial communities

Keywords: Betula pendula, Populus tremula, Populus nigra, Salix purpurea, native trees, Illumina MiSeq

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Pioneer trees of Betula pendula at a red gypsum landfill harbour specific structure and composition of root-associated microbial communities

Vanessa Alvarez-Lopez, Cyril Zappelini, Alexis Durand, Michel

Introduction

Gentle Remediation Options (GRO) focus on the use of plants in combination with associated microorganisms for the remediation and revitalisation of contaminated sites [START_REF] Kidd | Agronomic practices for improving gentle remediation of trace elementcontaminated soils[END_REF]. Metal(loid) toxicity can severely limit the performance and establishment of plants in polluted areas and therefore, the selection of metal-tolerant plant species able to successfully establish and grow at each specific site is of vital importance for the success of GRO. Sites contaminated with potentially toxic elements (PTE); such as mine tailings, spoils or landfills (made-up of by-products of metal extraction) are important sources of metal-tolerant plant genotypes. Red gypsum landfills are made of by-products of titanium refining; mainly used for the TiO2 pigment industry and enriched in S, Fe and Mn [START_REF] Zappelini | Streptomyces dominate the soil under Betula trees that have naturally colonized a red gypsum landfill[END_REF][START_REF] Zapata-Carbonell | Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity[END_REF].

Despite the unfavourable growth conditions at these sites, several metallophyte plants have evolved mechanisms to tolerate these conditions and are able to colonise these types of substrates [START_REF] Whiting | Research priorities for conservation of metallophyte biodiversity and their potential for restoration and site remediation[END_REF][START_REF] Batty | The potential importance of mine sites for biodiversity[END_REF][START_REF] Mendez | Phytostabilization of mine tailings in arid and semiarid environments-an emerging remediation technology[END_REF]. Fast-growing trees are ideal plants for the revegetation of these sites due to their extensive root systems, rapid growth, large biomass production and easy harvesting with subsequent re-sprouting [START_REF] Peuke | Phytoremediation[END_REF].

In the last years, an increasing number of studies including the use of native pioneer trees for the revegetation of degraded lands is found in the (Fernandez et al., 2017;[START_REF] Mleczek | Phytoextraction of potentially toxic elements by six tree species growing on hazardous mining sludge[END_REF]Asension et al., 2018) A greater understanding of the mechanisms, which influence the response of plants to the local conditions frequently found at these sites, could help to improve the efficiency of GRO techniques. Although an increasing number of studies have addressed the influence of the associated microbial community in plant metal tolerance, its role in harsh environments remains unravelled.

Studies approaching the description of plant-associated microbial communities are useful as a first approximation to understand these interactions. Plants interact closely with microbes, and these can enhance plant growth and health by increasing nutrient uptake and improving plant resistance to pathogens and stress [START_REF] Weyens | Exploiting plantmicrobe partnerships to improve biomass production and remediation[END_REF][START_REF] Kidd | Chapter Three -Potential Role of plant-associated bacteria in plant metal uptake and implications in phytotechnologies[END_REF]. Plant growth-promoting microorganismsinclude nitrogen-fixing bacteria, biocontrol microorganisms or mycorrhizal fungi, among others [START_REF] Coninx | Chapter Four -Mycorrhiza-Assisted Phytoremediation[END_REF][START_REF] Kidd | Chapter Three -Potential Role of plant-associated bacteria in plant metal uptake and implications in phytotechnologies[END_REF]. Plant associated microorganisms can also improve nutrient availability throughthe production of organic acids and siderophores or phosphate solubilisation.

Bacteria can also produce phytohormones, such as auxins, cytokinins and gibberellins, which stimulate plant growth and development [START_REF] Tanimoto | Regulation of root growth by plant hormones-roles for auxin and gibberellin[END_REF][START_REF] Taghavi | Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees[END_REF].

Plant associated bacteria can also reduce plant stress through the synthesis of 1aminocyclopropane-1-carboxylic acid (ACC) deaminase which reduces the level of ethylene in stressed plants (Glick, 2014). Other well-known mechanisms of PGPBs include increasing plant water uptake, alteration of root morphology, production of antibiotics and the induction of plant defence mechanisms [START_REF] Van Loon | Signalling in Rhizobacteria-Plant Interactions[END_REF][START_REF] Kidd | Trace element behaviour at the root-soil interface: Implications in phytoremediation[END_REF].

Ectomycorrhizal fungal (ECM) are the dominant group of root symbionts of pioneer trees that attempt to colonise heavily contaminated sites [START_REF] Colpaert | Chapter 11 Heavy metal pollution and genetic adaptations in ectomycorrhizal fungi[END_REF]. Fungal endophytes also play a key role on primary colonisation and subsequently on phytoremediation. It has been observed on contaminated sites that pioneer populations of mycorrhizal birches, willows, poplars or pines are able to successfully establish [START_REF] Vrålstad | Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats[END_REF][START_REF] Adriaensen | A zincadapted fungus protects pines from zinc stress[END_REF][START_REF] Colpaert | Evolutionary adaptation to Zn toxicity in populations of Suilloid fungi[END_REF]. Root-associated arbuscular mycorrhizal fungi (AMF) usually improve the growth of plants in non-fertile soils by increasing the exchange surface between plant and soil [START_REF] Smith | 5 -Mineral nutrition, heavy metal accumulation and water relations of VA mycorrhizal plants[END_REF]. AMF have been mostly studied for their role in plant growth, mainly due to their role in plant P uptake from soils [START_REF] Smith | 5 -Mineral nutrition, heavy metal accumulation and water relations of VA mycorrhizal plants[END_REF]. Moreover, AMF are able to alleviate metal toxicity in plants [START_REF] Hildebrandt | Arbuscular mycorrhiza and heavy metal tolerance[END_REF]. Other key soil microorganisms such as saprotrophic fungi are central in soils due to their capacity to degrade recalcitrant organic compounds and may strongly interact with soil bacteria [START_REF] Baldrian | Wood-inhabiting ligninolytic basidiomycetes in soils: Ecology and constraints for applicability in bioremediation[END_REF]. Saprotrophic fungi may also have a potential in mobilising metals from contaminated soils, due to their ability to exudate chelating agents [START_REF] Arwidsson | Remediation of metal contaminated soil by organic metabolites from fungi I-Production of organic acids[END_REF].

Further interactions between bacteria, fungi and plants have been shown to contribute to microbial community stability [START_REF] Bonfante | Plants, Mycorrhizal Fungi, and Bacteria: A Network of Interactions[END_REF][START_REF] Bell | Linkage between bacterial and fungal rhizosphere communities in hydrocarboncontaminated soils is related to plant phylogeny[END_REF] and many bacterial strains have indeed been reported to promote mycorrhizal symbioses (Frey-Klett et al., 2007;[START_REF] Hrynkiewicz | Ectomycorrhiza formation and willow growth promotion as affected by associated bacteria: role of microbial metabolites and use of C sources[END_REF].

Here, we characterised the root microbial communities associated to Betula pendula, which is the pioneer colonising vegetation at a red gypsum landfill in the region of Thann in NE France [START_REF] Zapata-Carbonell | Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity[END_REF]. At this site, B. pendula is the only woody species that had successfully established across the whole landfill while other woody species showed only sparse growth at some specific spots [START_REF] Zapata-Carbonell | Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity[END_REF]. Moreover, in previous studies B. pendula trees growing at this site have been shown to efficiently accumulate Mn and therefore this species is a promising candidate to be used as biocatalyst. The present study hypothesised that B. pendula hosts a specific root-associated microbial community responsible for its high adaptability to the soil conditions at this site. Therefore, the objective of this research was to analyse the structure of these communities using high-throughput sequencing technologies (Illumina nextgeneration sequencing).

Material and methods

Study site

The study site is located in the southern part of the Alsace region (47°47'47.7"N 7°08'18.5"E) near the city of Thann (NE of France). Here, Cristal France produces TiO2 (an important pigment) and TiCl4 (an excellent Lewis acid catalyst). The Thann plant produces TiO2 through a sulphate-based technology and TiCl4 through a chlorine process leading to the production of acidic effluents with a high load in trace metals. The extractive industry led to the production of by-products, such as red gypsum that are stored on large and open landfills. Red gypsum is produced by the neutralisation of wastewaters with limestone and is characterised by high Mn and Fe concentrations and elevated pH [START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF]. Betula pendula was found to be the dominant (> 80%) plant species that had naturally recolonised the D1 landfill area after the deposit of red gypsum ceased in 2006 [START_REF] Zapata-Carbonell | Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity[END_REF]. According to the "National methodology for the management of polluted sites and soils", (2017) developed by the French Ministry of Environment, Energy and Sea, and based on the data recorded by [START_REF] Zapata-Carbonell | Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity[END_REF], the D1 area presents values higher than those found in natural soils for Cd, Co, Cr, Cu, Pb, Zn and (especially) for Ti.

Sampling procedure

All soil and plant samplings were performed in October 2016 in the D1 landfill area (described previously by [START_REF] Zapata-Carbonell | Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity[END_REF]. Roots and shoots of B. pendula were collected across the D1 area. Ten individual plants of B. pendula were sampled at the centre of the landfill where B. pendula is the only tree growing. One soil sample was taken from the root system of each plant. For comparative purposes, 10 individual trees of other woody species growing at the edges (Populus tremula, P. nigra, Salix purpurea) were sampled at three specific spots. Moreover, 10 individual plants of B. pendula, which were found growing in close proximity to these species at the edges, were also sampled. These plant combinations were always growing at the same point and only one soil sample was taken for each pair of woody species. Soil samples (although not rhizospheric soils) were collected in the close proximity of the roots. To clarify, we named B. pendula associated to P. tremula as B. pendula-(Pt), those associated to P. nigra as B. pendula-(Pn) and those associated to S. purpurea as B. pendula-(Sp). Birch plants sampled at the centre were named as B. pendula (Fig. 1).

Soil physicochemical properties and elemental composition of leaves

Soil pH and extractable-element concentrations were determined in the < 2mm fraction of the dried soils. Soil pH was measured in H2O using a 1:2.5 soil:solution ratio after 30 min agitation. To determine the extractable fractions of soil macro-and micro-elements, 5 g of 2 mm sieved soil were incubated with 50 mL of 10 mM CaCl2 under agitation for 3 h at room temperature. The mixture was filtered with ash-free filters and acidified at 2% (v/v) with HNO3 before inductively coupled plasma atomic emission spectrometry analysis (ICP-AES, Thermo Fischer Scientific, Inc., Pittsburgh, PA, United States).

Plants were thoroughly washed with pressurised tap water to remove any adhering soil particles, and rinsed with deionised water. Leaves were dried for 48 h at 40 °C and 0.1 g of dry material was digested in a 4:1 HNO3:H2O2 mixture. Shoot nutrients and trace elements were measured also by ICP-AES.

Molecular methods

The roots were carefully washed with tap water to remove visible soil and rinsed with sterile deionised water. The fine roots were then cut off and transferred to Eppendorf tubes and frozen at -20 °C. DNA was extracted from roots of 10 single individual trees from each plant species at each sampling point. To isolate DNA from the roots, plant cell lysis was first performed using 50 mg of frozen root samples that were pulverised with 3mm tungsten carbide beads (Qiagen S.A.S., Courtaboeuf, France) in a Mixer Mill for 3 min at 30 Hz (modelMM400; Retsch Inc., Newtown, PA). Root DNA was then extracted with a PowerSoil® DNA Isolation Kit (MO-BIO Laboratories, Inc., Carlsbad, CA USA).

To improve DNA extraction, an additional step of 10 min at 60ºC was realised after addition of C1 solution. The DNA quality and quantity were assessed by agarose gel electrophoresis and with the Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA) using an FLX-Xenius spectrofluorometer (SAFAS, Monaco). Twostep PCR amplifications were performed with the bacterial primers 341F (5′-CCT ACG GGR SGC AGC AG -3′) and 802R (5′-GAC TAC HVG GGT ATC TAA TCC -3′) [START_REF] Baker | Review and re-analysis of domain-specific 16S primers[END_REF][START_REF] Beckers | Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies[END_REF] and the fungal primers ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A -3′) and ITS2 (5′-GCT GCG TTC TTC ATC GAT GC -3′) [START_REF] White | 38 -Amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics[END_REF]Gardes, 1993). The cycling conditions for 16S rRNA gene amplification involved an initial 3 min denaturing step at 94°C followed by 30 cycles of 25 s at 94°C, 25 s at 43.6°C, and 1:30 min s at 72°C. The cycling conditions for ITS genes included an initial 3 min denaturing step at 94°C followed by 30 cycles of 25 s at 94°C, 25 s at 45.4°C, and 1:30 min s at 72°C. The sequencing of the V3-V4 domains of 16S rRNA genes and the fungal ITS1 was performed with an Illumina MiSeq platform (Microsynth AG, Switzerland).

Data analysis

Bioinformatics processing of the bacterial datasets was carried out using the MOTHUR pipeline [START_REF] Schloss | Introducing mothur: Open-Source, platform-independent, community-supported software for describing and comparing microbial communities[END_REF]. Raw reads were filtered by length and quality and 16S rRNA gene reads were pre-clustered using sumaclust [START_REF] Mercier | Use of endophytic and rhizosphere bacteria to improve phytoremediation of arsenic-contaminated industrial soils by autochthonous Betula celtiberica[END_REF] at 0.99 identity. Sequences with less than 8 reads were discarded, then remaining sequences were aligned with those present in the Silva database to remove non-16S rRNA gene sequences, and plant DNA contamination was removed by suppressing reads identified as k__Bacteria (100), p__Cyanobacteria (100), c__Chloroplast (100), o__Streptophyta (100), and unclassified (100). Taxonomic assignments were made using a Bayesian approach [START_REF] Wang | Naïve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[END_REF] with the Greengenes database (DeSantis et al., 2006).

Finally, OTUs classification was derived using the Needleman distance and average neighbour clustering at a distance of 0.03.

Fungal Sequence de-multiplexing and bioinformatics processing of the datasets were performed using the PIPITS pipeline (Gweon et al., 2015). This pipeline uses the most recent RDP Classifier to classify sequences against the curated UNITE fungal dataset. As for bacteria, fungal sequences were filtered by length and quality and clustered into OTUs, defined at 97% similarity. Singleton OTUs were excluded (Dickie, 2010;[START_REF] Tedersoo | Ectomycorrhizal lifestyle in fungi: global diversity, distribution, and evolution of phylogenetic lineages[END_REF] and the taxonomic assignment of OTUs was performed using the UNITE [START_REF] Kõljalg | Towards a unified paradigm for sequence-based identification of fungi[END_REF] database at a 97% similarity threshold.

Statistical analysis

All statistical analyses were performed using R software v. 3.0.2 (R Core Team, 2013).

The Shapiro test was used to verify the normality of the data. Analysis of variance (ANOVA) was used to detect significant differences in shoot and soil measured parameters.

Richness and diversity indices were calculated based on a data set in which the numbers of reads per sample were rarefied to 6972 and 28129 reads per sample for bacteria and fungi, respectively. Rarefaction curves were created with the "rarecurve" function in the Vegan package. NMDS was calculated using the Bray Curtis method (k = 3) using the "metaMDS" function in the Vegan package. Results of relative abundance of the most abundant OTUs were paired with a heatmap of Spearman's correlations created with the "heatmap.2" function using the gplot package. The numbers of OTUs that were shared between plants were visualised using Venn diagrams implemented by "VennDiagram" in the Vegan package. Indicator species analysis was performed using the "multipat" function of the indicspecies package (De Caceres and Legendre, 2009). An ANalysis Of SIMilarities (ANOSIM) was carried out to obtain P (significance levels) and R (the strength of the factors on the samples) values using the "anosim" function with the vegan package. A spearman correlation was carried out to determine the relationships between soil/plant physicochemical characteristics and the abundant OTUs and between fungal and bacterial OTUs. Differences in family and OTUs abundance between plants were analysed using the Kruskal-Wallis test.

Results

Soil physicochemical properties and elemental composition of leaves

Supporting Information Table S1 and Supporting Information Table S2 present the main soil properties as well as the leaf macro-and micro-nutrients used in subsequent statistical analysis as environmental variables. In the case of trees growing on the edges, only one soil sample for each pair of plant species has been studied. Briefly, soils present a strongly alkaline pH (between 7.8 up to 10). The main CaCl2-extractable elements found at the landfill were Mg > K > Fe > Mn > P > Cu and a relation between soil pH and CaCl2extractable Mn and Fe was clearly observed. Supporting Information Table S2 evidences the Mn accumulation capacity of B. pendula, showing the highest levels of Mn leaf concentration (up to 570 mg kg -1 ). Moreover, this accumulation was not related to the site of growth (neither to the pH nor to the CaCl2-extractable fraction of Mn) (Table S1 andTable S2).Plants of birch also tended to accumulate a higher P concentration in their leaves than the other plant species growing at the same sampling points although this difference was not significant (Table S2).

Richness and diversity of root bacterial and fungal communities for the 4 woody species

Following total genomic DNA extraction from root samples, amplicons of the V3-V4 16S rRNA gene region were generated and a total of 5,262,333 paired-end reads were obtained through Illumina MiSeq sequencing (Supporting Information Table S3). After carrying out a quality, chimera, singleton and contaminant filter, a total of 907,006 filtered and non-chimeric bacterial sequences constituted our final processed dataset, spread among 1,325 non-singleton OTUs defined by representative DNA sequences with sizes of 231 to 257 bp (mean = 237 bp). After subsampling, the dataset was rarefied to 6,972 reads per sample (among the samples from each tree, those exhibiting a low sequence count were eliminated from the rest of the analysis), which were distributed among 1,322 nonsingletons OTUs. Amplicons of the ITS region were generated and a total of 5,581,697 paired-end reads were obtained (Supporting Information Table S4). A total of 5,148,296 filtered and non-chimeric fungal sequences constituted the final processed dataset, spread among 613 non-singleton OTUs defined DNA sequences with sizes above to 100 bp.

After subsampling, the dataset contained 28,129 reads per sample, distributed among 577 non-singletons OTUs.

Rarefaction curve analysis showed that all samples approached an asymptote in both the bacterial and fungal dataset, revealing that the overall diversity was well represented (Supporting Information Fig. S1). Moreover, the measured Good's coverage values (an estimator of completeness of sampling) were greater than 98% for each sample type in the case of the bacterial dataset, and 100% for the fungal dataset (Supporting Information Table S3 andS4 respectively).

Richness and diversity estimations were calculated for each dataset (Table 1 andTable 2). Both bacterial richness (based on Chao1 estimator and number of observed OTUs) and diversity indices (estimated through the Shannon Index, Inverse Simpson Index and Evenness) were generally lower in the roots of the B. pendula plants collected at the centre of the landfill than in the roots of all other trees collected at the edges. The highest bacterial richness and diversity indices were found to be associated with P. tremula (Table 1). Fungal richness and diversity (Table 2) followed the same trend as for bacteria and were generally lower in the roots of the B. pendula growing at the centre of the landfill. In this case, both richness indices (Chao1 and number of observed OTUs) were highest in the roots of all birches growing at the edges.

Effects of plant species and growth location in microbial structure and composition

The bacterial dataset comprised a total of 17 phyla, 151 families, and 246 genera (17, 79 and 100 taxonomically identified respectively). The bacterial community was dominated by Proteobacteria (38%), Actinobacteria (35%) and Bacteroidetes (20%). Only 26 (20 identified) families and 31 (14 identified) genera presented an abundance of higher than 0.5%. The fungal dataset was composed of a total of 7 phyla, 138 families, and 211 genera (5, 98 and128 taxonomically identified respectively). The fungal community was dominated by Ascomycota (60%) followed by Basidiomycota (30%). Only 18 (4 identified) families and 19 (5 identified) genera presented an abundance higher than 0.5%.

Figure 2 shows a barplot for the 20 most abundant bacterial (Fig. 2a) and fungal (Fig. 2b) families for each studied tree. The three most abundant identified families were Cytophagaceae (19%), Streptomycetaceae (16%) and Hyphomicrobiaceae (15%) (Fig. 2a) and the three most abundant identified genera were Lentzea (7.11%), Devosia (5.52%) and Rhodoplanes (3.33%) (data not shown). The roots of B. pendula showed a slight enrichment in members belonging to the genera Kribella, Rhodoplanes and Methylibium compared to the other trees (data not shown). The most abundant identified fungal family corresponded to Pyronemataceae (33%), followed by Russulaceae (15.5%). The next identified family was Thelephoraceae, although present in a much lower abundance (2.0%) (Fig. 2b). The most abundant genera were Geopora (33.1%), Lactarius (15.1%) and Tomentella (2.0%). Roots of B. pendula showed a slight increase in members of the genera Oidiodendron and Geopora (data not shown).

Figure 2 shows a clear pattern in the distribution of families between each pair of neighbouring trees (P. tremula -B. pendula (Pt), P. nigra -B. pendula (Pn) and S. purpurea -B. pendula (Sp)). However, due to the high data scattering, few of these results were significant. In the case of the bacterial dataset, the statistical differences were mainly associated to P. nigra and the neighbour birch (named as B. pendula (Pn)). These trees harboured a higher number of members of the families Actinosynnemataceae (Kruskal Wallis χ2=37.9, p=1.2×10 -6 ), Glycomycetaceae (Kruskal Wallis χ2=34.61, p=5.2×10 -6 ) and Comamonadaceae (Kruskal Wallis χ2=20.6, p=2.1×10 -3 ) and lower members of the families Pseudonocardiaceae (Kruskal Wallis χ2=32.4, p=1.4×10 -5 ) and Methylocystaceae (Kruskal Wallis χ2=39.2, p=6.6×10 -7 ) than all the other trees. In the case of the fungal dataset the statistical differences were observed only for two families Pyronemataceae (Kruskal Wallis χ2=24.4, p=4.4×10 -4 ) and Sebacinaceae (Kruskal Wallis χ2=20.5, p=2.3×10 -3 ), which showed respectively a lower and higher number in roots associated to P. tremula and the nearby growing birch (B. pendula (Pt))

Table 3 shows the result of the ANOSIM analysis for the dissimilarity values of microbial communities between samples. A significant dissimilarity (p < 0.05) was observed when all the tree species growing at the landfill were analysed together.

Interestingly, when comparing B. pendula from the centre of the landfill with the other tree species (Salix and Populus) or with the other B. pendula growing at the edges, a strong and significant dissimilarity in their root-associated bacterial communities was observed. Similarly, trees growing at the different sampling points at the edges showed a significant dissimilarity amongst their associated microbial communities. However, trees of B. pendula growing at the edges of the landfill (B. pendula (Pt), B. pendula (Pn) and B. pendula (Sp)) did not show a significant dissimilarity between their root bacterial communities and those of their neighbour trees (P. tremula, P. nigra and S. purpurea respectively).

The dissimilarity analysis between different root fungal communities, (ANOSIM, Table 3) showed similar patterns as for bacteria although less marked. The highest dissimilarity was found between B. pendula in the centre with either B. pendula (Pt) (R 2 =0.621, p=0.001) or with P. tremula (R 2 =0.804, p=0.001). A high and significant dissimilarity was found also when comparing together (a) all the plants of B. pendula growing at the different locations (R 2 =0.327, p=0.001); (b) the B. pendula at the centre and the other tree species (R 2 =0.471 p=0.001), (c) the other tree species growing at the edges (either when comparing only B. pendula (R 2 =0.327, p=0.001) or (d) all species (R 2 =0.472 p=0.001)). No significant dissimilarity was observed when comparing B. pendula either with B. pendula (Pn) or with P. nigra. S5 shows the number of total reads of the most abundant OTUs (>0.5%) found in the bacterial collection. Only 33 OTUs (from the 1,322) presented abundances higher than 0.5%, highlighting the presence of very rare OTUs dominating the community. The most abundant OTU belongs to the family Streptomycetaceae (OTU00001, 14.9%). This OTU presented the highest abundance in roots of B. pendula and S. purpurea, although it was also uniformly distributed in the roots of all the plant species and locations. Within the other abundant OTUs, OTU00005_f:Micromonosporaceae, OTU00022_g:Kribella, OTU00030_f:Methylocystaceae and OTU00034_g:Methylibium tended to be more abundant in roots of B. pendula growing at the centre of the landfill (although significant differences were not observed).

Supporting Information Table

Total nº of reads of the most abundant OTUs (>0.5%) in the fungal dataset are shown in Supporting Information Table S6. Similar to what was observed for bacteria, only few OTUs presented abundance >0.5% (24 from a total of 577). The fungal community was clearly dominated by the OTU 25_92637 belonging to Geopora arenicola species (31.8% of the total dataset). This OTU was present in high abundance in roots of all the studied trees, however, its abundance was slightly higher in the roots of B. pendula at the centre. An extreme variability between replicates of the same plant species or growth location (which is reflected by the high standard errors in Supporting Information Table S6) was observed, leading to the lack of important statistical differences. The other most abundant OTUs belonged to the genera Lactarius (15.7%) and the order Pezizales (7.2%), Agaricales (5.7%) and Helotiales (3.6%).

Supporting Information Figure S2 shows the distribution of unique and shared bacterial (Supporting Information Fig. S2a

Relationships between microbial communities, physicochemical soil properties and elemental composition of leaves.

Considering an interspecies analysis based on the bacterial and fungal community composition using the most abundant OTUs (>0.5%), (NMDS plots (Fig. 4)) it was observed that roots of B. pendula, P. tremula, P. nigra and S. purpurea were clustered separately for each tree. The distribution of the samples of B. pendula was related to the abundance of 5 specific bacterial OTUs (Otu00001 (f:Streptomycetaceae), Otu00017

(g:Rhodoplanes), Otu00009 (f:Cytophagaceae), Otu00030 (f:Methylocystaceae) and Otu00014 (f:Haliangiaceae)) and to leaf Mn-concentration.

Supporting Information Table S7 shows the one-by-one correlations found between the most abundant bacterial OTUs (>0.5%), soil physicochemical parameters and leaf metal and nutrient concentrations. On the whole, correlations of several OTUs with both, CaCl2-extractable Fe and Mn concentrations were observed (either positive in the case of OTU00003_g:Lentzea, OTU00006_g:Devosia, OTU00012_g:Glycomyces, and OTU00029_g:Catellatospora or negative in OTU00001 and OTU00026 both belonging to the Streptomycetaceae family). Also strong positive correlations were found with some soil nutrients, such as CaCl2-extractable P (OTU00002_f:Cytophagaceae, OTU00026_f:Streptomycetaceae and OTU00028_g:Arenimonas) or CaCl2-extractable K (OTU00005_f:Micromonosporaceae, OTU00016_o:Rhizobiales or OTU00037_g:Dyadobacter). However, correlations with the corresponding leaf nutrient concentrations were not observed (except in the case of OTU00002_f:Cytophagaceae where a correlation was observed with soil CaCl2-extractable P and leaf P concentration).

A positive correlation with both leaf K and P concentrations was found with OTU00003_g:Lentzea and OTU00010_f:Cytophagaceae. Finally, OTU00012_g:Glycomyces showed a positive correlation with leaf P and OTU00029 with leaf K concentrations. Similarly, in the case of fungal community, a strong correlation with both, CaCl2-extractable Fe and Mn was observed for the OTUs 24_66088_o:Pezizales, 23_37852_o:Agaricales and 51_62345_Unclassified. The OTU 24_66088 also showed a correlation with leaf K and P concentrations while the OTU 67_38353_o:Hypocreales was correlated with Mn leaf concentrations and OTU 51_62345_Unclassified with leaf P concentrations (Supporting Information Table S8).

Finally, a spearman correlation was carried out to study significant co-occurrence in the community of the most abundant bacterial and fungal OTUs. A positive cooccurrence may be considered as co-presence while a negative co-occurrence as mutual exclusion (Supporting Information Table S9).

Discussion

This study aimed to describe the root-associated microbiota of B. pendula which naturally colonises a red-gypsum landfill in the NW of France. It is well known that birch is a pioneering plant species, able to establish in nutrient deficient and degraded soils [START_REF] Kopponen | Clonal differences in copper and zinc tolerance of birch in metal-supplemented soils[END_REF]. Part of this success is related to their well-developed root system( Birch is also a high biomass producer and fast-growing tree which can provide high economic return with end-use applications such as the production of biofuels, pulp, and paper and other bio-based products such as biocatalysts [START_REF] Kidd | Agronomic practices for improving gentle remediation of trace elementcontaminated soils[END_REF][START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF]. Furthermore, birch can be grown on marginal land thus evading the food versus fuel debate [START_REF] Searchinger | Use of U.S. croplands for biofuels increases greenhouse gases through emissions from land-use change[END_REF]. On the other hand, plants harbour multiple microbial taxa, which affect several plant traits such as biomass production, nutrient availability and transport from the soil [START_REF] Sugiyama | Relationships between Arabidopsis genotype-specific biomass accumulation and associated soil microbial communities[END_REF], metabolite production [START_REF] Badri | Potential impact of soil microbiomes on the leaf metabolome and on herbivore feeding behavior[END_REF], drought tolerance [START_REF] Lau | Rapid responses of soil microorganisms improve plant fitness in novel environments[END_REF] and flowering time [START_REF] Wagner | Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative[END_REF]. In the last years, several studies have also highlighted the influence of mycorrhizas on the alleviation on metal toxicity to plants through extracellular inactivation, metal binding in fungal wall, enhanced efflux pumps activity, intracellular inactivation and adaptive response to oxidative stress [START_REF] Amir | Role of Mycorrhizal Fungi in the Alleviation of Heavy Metal Toxicity in Plants[END_REF]). In addition, the composition of the plant microbiota can enhance host resistance to pathogens [START_REF] Mendes | Deciphering the rhizosphere microbiome for disease-suppressive bacteria[END_REF][START_REF] Busby | Common foliar fungi of Populus trichocarpa modify Melampsora rust disease severity[END_REF].

Little information about root microbial communities associated with B. pendula growing at contaminated sites using metabarcoding approaches is available in the Here, B. pendula growing at the centre of the landfill showed, generally, lower richness and diversity (either in bacterial and fungal communities) than the other trees growing at the edges. One possible explanation could be the presence of harsher physicochemical soil properties at the centre of the landfill, which would negatively affect root microbial communities. However, we did not find any clear difference in the measured parameters (pH and CaCl2-extractable elements) between the different studied areas. Similarly, in a deeper study of the area, Zapata-Carbonell et al. (2019) did not observe any pattern of distribution on measured soil nutritional parameters (based on organic matter and CaCl2-extractable elements) between the centre and the edges of the landfill. However, it is also important to note that other factors (such as water, N content or CEC) could also be affecting the distribution and composition of plant and microbial communities. It has been reported by several authors that beneficial microorganisms, increase plant tolerance to drought, , [START_REF] Timmusk | Drought-Tolerance of Wheat Improved by Rhizosphere Bacteria from Harsh Environments: Enhanced Biomass Production and Reduced Emissions of Stress Volatiles[END_REF]. Moreover, the incidence and virulence of diseases or pests can be higher in plants growing in stressed environments (such as degraded soils). Associated microorganisms can also protect plants against diseases through for example the induction of plant defence mechanisms or the production of antibiotics [START_REF] Compant | Plant growth-promoting bacteria in the rhizo-and endosphere of plants: Their role, colonization, mechanisms involved and prospects for utilization[END_REF]. Related to that point, it is worth mentioning the importance of members of the order Actinomycetales (notably the Streptomyces genus), as the richest natural source of antibiotics or antimetabolites [START_REF] Bérdy | Bioactive Microbial Metabolites[END_REF][START_REF] Olano | Antitumor compounds from marine actinomycetes[END_REF][START_REF] Lemanceau | Contribution of studies on suppressive soils to the identification of bacterial biocontrol agents and to the knowledge of their modes of action[END_REF].

In the present study, the bacterial community was dominated by Proteobacteria and Actinobacteria. It is important to note that here, no distinction between endophytes and epiphytes was made, and therefore only one habitat was considered. However, roots were exhaustively washed and we can consider that only strongly root-associated microorganisms are being discussed. Interestingly, Proteobacteria (families such as Pseudomonadaceae, Burkholderiaceae or Xanthomonadales) and several OTUs belonging to Actinobacteria have been shown to be the most dynamic taxa associated with disease suppression in the rhizosphere of sugar beet plants in a study using DNA metagenomics with Phylochip [START_REF] Mendes | Deciphering the rhizosphere microbiome for disease-suppressive bacteria[END_REF].

Mesa et al. (2017) also found that in the endosphere of B. celtiberica, Proteobacteria was the most abundant phylum (accounting for 64% of the total reads), followed by Bacteroidetes (17%), Actinobacteria (7.9%), Firmicutes (1.8%), and

Chlamydiae (1.3%). It is generally accepted that the Alphaand Betaproteobacteria are copiotrophic members (Fierer et al., 2007;[START_REF] Zhao | Responses of bacterial communities in arable soils in a rice-wheat cropping system to different fertilizer regimes and sampling times. PloS one 9:e85301 Figure 3 Heat map and hierarchical cluster analysis of the most abundant bacterial and fungal OTUs (>0.5%). The dendrogram represents linkage clustering using Euclidean distance measures. OTU delineation was based on a threshold of <97% sequence similarity. Assignments between brackets show the lowest taxonomic level associated with the OUT: k kingdom, p phylum, o order, c class, f family, g genus, s genus_species B[END_REF], since they are usually associated with habitats having relatively low soil C/N ratio, high NO-3-N [START_REF] Nugroho | Presence of Nitrosospira cluster 2 bacteria corresponds to N transformation rates in nine acid Scots pine forest soils[END_REF], and enriched nutrients [START_REF] Leff | Consistent responses of soil microbial communities to elevated nutrient inputs in grasslands across the globe[END_REF][START_REF] Li | Responses of soil bacterial communities to nitrogen deposition and precipitation increment are closely linked with aboveground community variation[END_REF]. Roots constitute a nutrient-rich niche since they receive high amounts of available C and N as exudates, which could promote the growth of these bacterial inside the root and at the rhizoplane. Otu01204 and Otu00214)) with an abundance ranging from 0.001% to 0.064%.

Moreover, OTUs belonging to the order Rhizobiales (including members of the family Hyphomicrobiaceae and the genera Rhodoplanes) were found to be indicators of the roots of B. pendula. There is little information about the role of Rhodoplanes in the environment, and several studies related their preference to inhabiting rhizospheric soil rather than endospheric habitats [START_REF] Touceda-González | Combined amendment of immobilizers and the plant growth-promoting strain Burkholderia phytofirmans PsJN favours plant growth and reduces heavy metal uptake[END_REF]Gkarmiri et al., 2017).

These bacteria were also abundant in the root DNA-based community of Arabidopsis thaliana (Haichar et al., 2012) and Panax notoginseng [START_REF] Tan | Diversity and composition of rhizospheric soil and root endogenous bacteria in Panax notoginseng during continuous cropping practices[END_REF]. A study which used 15 N-DNA-SIP to investigate soil microorganisms responsible for N fixation, identified, among others, bacteria of the genus Rhodoplanes as being potential N fixers [START_REF] Buckley | Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil[END_REF]. The abundance of microorganism related to N-cycling together with the low values of soil N reported in previous studies (Zapellini et al. 2018) seem to indicate that these bacteria could be important for the growth of birch in the landfill.

Several members of rhizobiales (such as Ensifer, Rhizobium, Phyllobacterium) have been shown to reduce plant stress response and improve plant growth in different hosts and soils [START_REF] Larcher | Bacteria from the rhizosphere and roots of Brassica napus influence its root growth promotion by Phyllobacterium brassicacearum[END_REF]Guo and Chi, 2014;[START_REF] Lafuente | Unraveling the effect of arsenic on the model Medicago-Ensifer interaction: a transcriptomic meta-analysis[END_REF]Mesa et al., 2017). Mesa et al. (2017) have also observed members of the Rhizobiales present in both the rhizosphere and endosphere of B. celtiberica. Moreover, in the same study, most of the culturable strains from the endosphere belonged to the genus Rhizobium (11%).

Actinobacteria was the second most represented phylum and the main OTU (Otu00001) of the dataset belonged to the Streptomycetaceae family. The predominance of this phylum was also found in several other studies involving either soil or plant compartments [START_REF] Lopez | Nickel drives bacterial community diversity in the rhizosphere of the hyperaccumulator Alyssum murale[END_REF]. For example, Ghosh and Das (2018) studied the soil bacterial community in Mn-enriched mine tailings by using a shotgun-metagenomic approach and found that Proteobacteria, and Actinobacteria dominated the soil community (and specifically the Streptomycetaceae family). Several studies have confirmed the high adaptability of Actinobacteria to toxic concentrations of metals (DeGrood et al., 2005;[START_REF] Schmidt | Heavy metal resistance mechanisms in actinobacteria for survival in AMD contaminated soils[END_REF] and it has been shown that their strong secondary metabolism enables them to cope with stress factors [START_REF] So | A lead-absorbing protein with superoxide dismutase activity from Streptomyces subrutilus[END_REF]. In one of our recent study [START_REF] Zappelini | Streptomyces dominate the soil under Betula trees that have naturally colonized a red gypsum landfill[END_REF], we found that Actinobacteria (and specifically the genera Streptomyces) was the predominant phylum in the soil culturable bacterial collection isolated from this same soil. The high proportion of the Actinobacteria phylum, which was found amongst all tree roots (independently of the species and growth location), could suggest the importance of the soil bacterial community in determining the structure and composition of root-associated communities. Similarly, Yang et al.

(2017) found that after growing barley seeds in soils, an enrichment in Actinobacteria was observed in roots of plantlets compared to seeds growing in axenic substrate, suggesting that the soil had a major impact driving the root bacterial communities.

The biological control activity of these endophytic actinobacteria is also of interest given their ability to colonise healthy plant tissues and produce antibiotics in situ [START_REF] Shimizu | Endophytic actinomycetes: Biocontrol agents and growth promoters[END_REF]. For example, the endophytic Streptomycetes isolated from healthy banana plants (Musa sp.) were studied for the ability to produce antifungal molecules that inhibited the growth of the pathogen Fusarium oxysporum [START_REF] Cao | Isolation and characterization of endophytic streptomycete antagonists of fusarium wilt pathogen from surface-sterilized banana roots[END_REF]. An enrichment of Streptomyces in roots compared to rhizospheric soil is frequently described in the literature (Foulon et al., 2016;Durand et al., 2018). Moreover, Gkarmiri et al.

(2017) suggested that within the active bacterial community that Streptomyces were highly active in the root compartment but not in the rhizosphere.

In the present study, Otu00716 belonging to Catellatospora sp. (family Micromonosporaceae, phylum Actinobacteria) was found to be an indicator species of the birch trees at the central location. The genus Catellatospora is considered one of the rarely occurring genera of actinomycetes: only a few papers mentioning these microorganisms can be found and most of them concern descriptions of the few species and their taxonomic position [START_REF] Saracchi | Variability on Catellatospora spp. strains isolated from plant materials[END_REF]. During a study of the endophytic root actinomycete population from 252 leaf litter and 205 plant root samples [START_REF] Petrolini | A sporangiate actinomycete with unusual morphological features: Streptosporangium claviforme sp[END_REF][START_REF] Sardi | Isolation of endophytic Streptomyces strains from surface-sterilized roots[END_REF] found that most of the 2950 isolates obtained belonged to

Streptomyces [START_REF] Sardi | Isolation of endophytic Streptomyces strains from surface-sterilized roots[END_REF] and 215 to the genus Micromonospora [START_REF] Williams | Detection and identification of novel actinomycetes[END_REF]. However, representatives of the "rare" genera were also found [START_REF] Petrolini | A sporangiate actinomycete with unusual morphological features: Streptosporangium claviforme sp[END_REF][START_REF] Petrolini | Actinoplanes strains isolated from roots: morphological investigations[END_REF], and among these, 53 strains of Catellatospora sp. However, in the present study 6 OTUs belonging to this genus were observed. Moreover, one of these (Otu00029) was one of the abundant OTU (abundance >0.5%), although this was not the OTU with highest abundance in the birch roots.

Members of the plant microbiota can be transmitted either horizontally (acquired from the surrounding environment) or vertically (acquired directly from the parent) (Gundel et al., 2011). The results showed that B. pendula harboured a specific microbial community which is unique and different, not only from other plant species growing at the red gypsum landfill (Populus or Salix spp.) but also from other B. pendula growing at the edges (analysis of similarities ANOSIM, heatmap and hierarchical cluster analysis of the relative abundance of bacterial and fungal OTUs), suggesting that horizontally transmission is an important via of microbial acquisition in this case. Moreover, this plantlocation specific community was more noticeable in the case of the bacterial community.

However, concerning the distribution of unique and shared OTUs, it was found that, even though a high and consistent number of OTUs were unique for each plant species, most of them were shared among different plant species or sites of growth. These results also suggest that a generalised plant colonisation by certain indigenous soil bacterial species is occurring and that the specificity of microbial communities can be related mainly to the differences between OTU abundances. [START_REF] Yang | Dominant groups of potentially active bacteria shared by barley seeds become less abundant in root associated microbiome[END_REF] found that the differences in root active microbiomes associated with different barley cultivars were mainly quantitative and not as much qualitative. For example, the variation between the genotypes was manifested in the abundance of many OTUs from diverse taxa, rather than by the presence/absence of single OTUs in the given genotypes.

Data obtained from fungal sequencing showed a much higher replicate variability than those obtained in bacterial sequencing. The same pattern was found in several other studies when comparing fungal communities from soil and root habitats [START_REF] Beckers | Structural variability and niche differentiation in the rhizosphere and endosphere bacterial microbiome of fieldgrown poplar trees[END_REF]Durand et al., 2017;[START_REF] Kolaříková | Root-associated fungal communities along a primary succession on a mine spoil: Distinct ecological guilds assemble differently[END_REF]. This result suggests that fungal endophytic colonisation and formation of stable communities appears to be a more variable process than bacterial colonisation [START_REF] Beckers | Structural variability and niche differentiation in the rhizosphere and endosphere bacterial microbiome of fieldgrown poplar trees[END_REF].

Although in our study most taxa (of both bacterial and fungal communities but especially in the case of fungi sequencing) were not sequenced at a high enough read depth to enable powerful comparisons, an enrichment of the genus Geopora (specifically G. arenicola) in the roots of all tree species was observed and tended to be slightly higher in roots of B. pendula at the centre. [START_REF] Kolaříková | Root-associated fungal communities along a primary succession on a mine spoil: Distinct ecological guilds assemble differently[END_REF] investigated the rootassociated fungal communities of two dominant trees Salix caprea and B. pendula along a primary successional chronosequence on a mine spoil bank in the Czech Republic and found that G. arenicola) was specifically associated to the youngest trees in the chronosequence independently of the tree species. Geopora arenicola is an ectomycorrhizal fungus, which, among others has the characteristic to promote plant growth in stressful environments [START_REF] Hrynkiewicz | Promotion of mycorrhiza formation and growth of willows by the bacterial strain Sphingomonas sp. 23L on fly ash[END_REF]. Within the fungal indicator OTU, we found the genera Oidiodendron and Russula (OTU 3_5515 s:Oidiodendron_maius|SH217755.06FU and OTU 4_35502 g:Russula). Russula is an ECM fungus frequently found in soils and also associated with birch cultures [START_REF] Bent | Root-associated ectomycorrhizal fungi shared by various boreal forest seedlings naturally regenerating after a fire in interior Alaska and correlation of different fungi with host growth responses[END_REF][START_REF] Parker | Slowed biogeochemical cycling in sub-arctic birch forest linked to reduced mycorrhizal growth and community change after a defoliation event[END_REF]. Moreover, [START_REF] Parker | Slowed biogeochemical cycling in sub-arctic birch forest linked to reduced mycorrhizal growth and community change after a defoliation event[END_REF] screened plant/soil fungal communities associating with Picea mariana, Populus tremuloides and Betula papyrifera after a wildfire and found that each plant species seemed to be affected by different groups of fungi. A higher shoot biomass of birch was associated with those associated with ribotype 732 to 734 bp belonging to Russula spp. It is interesting to note, however, that the Russulaceae family was not abundant in roots of the central B. pendula. It seems that the specificity of the root-associated microbial community of the central birch plants is not related to the most abundant taxa. Interestingly, Durand et al. (2017) found that the less abundant fungal OTUs present a higher co-occurrence network connectivity than the most abundant OTUs. In the present study it was also observed that the most abundant OTUs (both fungal and bacterial) showed a low number of bacterial and fungal correlations. Fungi and bacteria share the same habitats and are therefore almost certain to frequently interact in soil and plants [START_REF] Wd | Living in a fungal world: impact of fungi on soil bacterial niche development[END_REF][START_REF] Zhao | Responses of bacterial communities in arable soils in a rice-wheat cropping system to different fertilizer regimes and sampling times. PloS one 9:e85301 Figure 3 Heat map and hierarchical cluster analysis of the most abundant bacterial and fungal OTUs (>0.5%). The dendrogram represents linkage clustering using Euclidean distance measures. OTU delineation was based on a threshold of <97% sequence similarity. Assignments between brackets show the lowest taxonomic level associated with the OUT: k kingdom, p phylum, o order, c class, f family, g genus, s genus_species B[END_REF]. Interactions between co-occurring bacteria and fungi play a considerable part in determining their respective roles, and investigating these interactions will lead to a more complete understanding of microbial ecology [START_REF] De Menezes | Linking fungal-bacterial co-occurrences to soil ecosystem function[END_REF].

Surprisingly, the second indicator OTU belonged to the ericoid mycorrhizal group

Oidiodendron maius [START_REF] Chambers | Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest[END_REF][START_REF] Lukešová | The potential of dark septate endophytes to form root symbioses with ectomycorrhizal and ericoid mycorrhizal middle european forest plants[END_REF]; although no Ericaceae plants are described at this landfill. Although less frequently, this mycorrhizal type has indeed been found in association with other plant species [START_REF] Chambers | Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest[END_REF] Similarly to what was observed in the bacterial dataset (although not clearly reflected by the ANOSIM analysis), B. pendula growing at the centre hosted a specific fungal community while trees growing at the edges shared an important part of both root community structure and composition. [START_REF] Bogar | New wrinkles in an old paradigm: neighborhood effects can modify the structure and specificity of Alnus-associated ectomycorrhizal fungal communities[END_REF] studied the relative importance of host and neighbourhood effects on the ECM fungal communities associated with Alnus rhombifolia (a host of specific ECM fungi) and Betula occidentalis (a host of generalist ECM fungi). The authors found that while the host identity acts as a primary filter on the composition and diversity of ECM fungal communities, proximity to a closely related host can mediate significant changes in community structure. In the same way, [START_REF] Hausmann | Plant neighborhood control of arbuscular mycorrhizal community composition[END_REF] found that the structure of arbuscular mycorrhizal (AM) fungal communities on neighbouring grass plants depended upon which pairs of host species were near each other. Neighbourhood effects can also be driven by changes in abiotic conditions that favour the growth of certain ECM fungal species, as demonstrated by [START_REF] Meinhardt | Disrupting mycorrhizal mutualisms: a potential mechanism by which exotic tamarisk outcompetes native cottonwoods[END_REF]; in that study, the authors examined the influence of invasive Tamarix spp. on the mycorrhizal associations of native Populus fremontii in an early-invasion field site and in the greenhouse and laboratory. They found that the ECM fungal community structure on P. fremontii shifted significantly in the presence of Tamarix spp., likely as a result of increases in soil nitrate concentrations and electrical conductivity associated with soils affected by Tamarix.

Some ECM fungi, Russula luteolus, Suillus bovinus, and Hebeloma crustuliniforme, form highly branched mycelial strands up to 40 cm [START_REF] Skinner | The uptake and translocation of phosphate by mycelial strands of pine mycorrhizas[END_REF]Finlay and Read, 1986a, b). In addition to the beneficial effects on the plant nutritive status, during the scavenging process fungi will simultaneously colonise more than one plant found at the site.

As previously described, abundances of both fungal and bacterial phyla were highly variable. Ghosh and Das (2018) also found a high variability in the abundance of different phyla and classes in the soil bacterial communities of a Mn-enriched tailing by using a shotgun-based approach. These authors suggest that this could be the response of heterogeneity in the environmental conditions and they highlight the importance of studying and integrating results of bacterial diversity and biotic and abiotic related factors for its correct interpretation. Although significant correlations were found between bacterial and fungi OTUs and soil physicochemical properties or plant nutrients, these were generally very weak (rho value usually below than 0.6). This could be due to the lack of a strong gradient in soil properties and rather a random heterogeneity found in these constructed waste landfills. Moreover, although this study involved the characterisation of epispheric root microorganisms (which would be directly influenced by soil properties) most of the analysed community belonged to the endophytic community and therefore are not directly influenced by the soil physicochemical properties. However, some interesting results were found, for example the correlation between some bacterial and fungal OTUs and both soil CaCl2-extractable Fe and Mn.

This could indicate the co-dissolution of Mn-and Fe-oxides from this soil leading to the release of both elements. A deeper study involving the isolation of these bacterial strains and characterisation of the biological pathways involved in this process would be interesting for further exploitation of these bacterial strains in Mn-biomining. [START_REF] Das | Manganese biomining: A review[END_REF] review the role of microorganisms in Mn-bioleaching operations for the recovery of metals from low-grade ores that would be otherwise unfeasible using current metallurgical technologies by their prohibitively high energy and capital costs. In general, dissolution of Mn-rich minerals occurs through enzymatic reduction which is often carried out by bacteria in the form of respiration where oxidised Mn acts as a terminal electron acceptor or by the indirect release of reductive compounds resulting from bacterial metabolism [START_REF] Das | Manganese biomining: A review[END_REF]Ghosh et al., 2016). In both cases the biological process occurs in the presence of organic carbon and energy sources [START_REF] Das | Manganese biomining: A review[END_REF]Ghosh et al., 2016). As it is well known, roots are a C-rich environment since up to 50 % of the net photosynthetic carbon is released from plant roots into the rhizosphere as rhizodeposits [START_REF] Curl | The Rhizosphere[END_REF]. At the same time, it is known that the molecular mechanisms behind Fe and Mn tolerance are frequently the same in bacteria [START_REF] Chandrangsu | Metal Homeostasis and Resistance in Bacteria[END_REF].

Finally, several OTUs were correlated with either soil nutrient availability and/or shoot nutrient concentrations. All trees growing at the area present shoot P and K deficiency (according to the values reported by [START_REF] Marschner | Mineral nutrition of higher plants[END_REF]), and therefore plantassociated microorganisms involved in the uptake and absorption of these elements can play a key role in the growth of the vegetation at the site. At the same time, [START_REF] Zapata-Carbonell | Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity[END_REF] found a correlation between the distribution of B. pendula at the site and CaCl2-extractable P and organic matter, highlighting again the importance of nutrients in the growth of the plants at this site.

Conclusions

To successfully implement a phytomanagement strategy it is vitally important to characterise the plant-soil-microbial system. In this study we characterised the microbial community associated with the roots of B. pendula, the most successful plant species colonising a red gypsum landfill. The bacterial community associated with birch growing in the centre of the landfill was very different from that of other tree species studied at the mine but also that of B. pendula growing at the edges of the landfill. The most abundant bacterial OTU (Otu 00001) belonged to the family Streptomycetaceae and was widespread through all the studied roots. At the same time, according to the NMDS analysis, this OTU seemed also to be related to the presence of birch at the centre of the landfill. Other bacterial OTUs associated to birch roots (such as members of the order Rhizobiales) are known to be involved in the N cycling, suggesting that this may be a critical factor for effective growth of the plants at the site.

Data obtained from fungal sequencing showed a much higher replicate variability than those obtained in bacterial sequencing. In the case of fungi, a less clear pattern was observed although it seems also that birch selected some specific OTUs (e.g OTUs belonging to the genera Oidiodendron and Russula, and the order Agaricales and Hypocreales were found to the indicators of the presence of B. pendula).

Several OTUs specifically associated to the birch growing at the centre of the area are known to be involved in nutrient cycling (e.g. members of Rhizobiales or the fungal species Geopora) and can therefore play a role in the high capacity of this plant to tolerate the growth limiting conditions in the landfill.

Differences between microbial communities (bacterial and fungal) were based on microbial structure but also on microbial composition and distribution: e.g. the Venn diagram showed that the unique bacterial and fungal OTUs associated to each plant were always present in a very low proportion in the total community (< 0.5% of abundance).

However, an analysis of the most abundant OTUs, generally shared by all plants (e.g. heatmap or NMDS) also showed a specificity of the communities associated with B.

pendula. Finally, it is also important to remember that assemblages of host-associated Only the 20 most abundant identified families are presented.

  and S2b) and fungal OTUs (Supporting Information Fig.S2c and S2d) amongst B. pendula growing at the centre and the other trees growing at the edge. B. pendula from the centre presented 44 specific OTUs compared to other tree species and 60 specific OTUs when compared to other birch trees growing at the edges. The number of specific OTUs in the B. pendula growing at the edges (compared to the B. pendula at the centre) was similar to that found in their neighbour trees. A similar pattern in the fungal dataset was found: B. pendula from the centre presented 5 specific OTUs when compared to the other B. pendula but also when compared to the other tree species.A heatmap was carried out using the most abundant bacterial and fungal OTUs (>0.5% abundance) (Fig.3). B. pendula growing at the centre clustered separately from all other trees. Trees of different species, which were growing together at the edges were clustered in couples, depending on their growth location. The birches at the centre of the landfill were more closely associated to trees growing at the locations of P. nigra. When a heatmap was performed separately for either bacterial or fungal communities (data not shown) the same pattern was found in the case of bacteria. Similarly, birch trees also presented a specific fungal OTU distribution but in this case, plants of B. pendula were associated to the cluster made up of P. tremula-B.pendula (Pt) and S. purpurea-B. pendula (Sp) (data not shown).An analysis of indicator OTUs for each tree species and growth locations was also carried out. A summary for B. pendula indicator OTUs is presented in Table4. Fourteen indicator bacterial OTUs and 4 indicator fungal OTUs were observed for this tree species.The bacterial OTUs include one belonging to the class Gammaproteobacteria, one to the family Cytophagales, 3 to the family Rhizobiales, one to the family Streptomycetaceae and one to the family Hyphomicrobiaceae, one to the genus Rhodoplanes and one to the genus Catellatospora. In the case of fungi, OTUs belonged to the species Oidiodendron maius, the genus Russula, and the order Agaricales and Hypocreales.

  presented the highest numbers of significant cooccurrence with fungal OTUs (either positive or negative). The fungal OTUs 44_70433_g:Lactarius, 24_66088_o:Pezizales and 18_28512_p:Ascomycota showed the highest number of significant co-occurrence with bacterial OTUs. On the contrary, ) which did not show any significant correlation with the occurrence of either fungal or bacterial OTUs, respectively. The most abundant fungal OTU 25_92637_s:Geopora arenicola presented a significant correlation with 5 bacterial OTUs and 4 of these were negative. The unique positive correlation was found was found to be significantly related to three fungal OTUs (from those positively correlated with 37_4836_c:Sordariomycetes and 3_31227_o:Agaricales). Overall, the strongest correlations (rho>0.5) were found between fungal OTU 24_66088_o:Pezizales and bacterial Otu00012_g:Glycomyces (rho=0.55), the fungal OTU 10_6322_Unclassified and the bacterial Otu00034_g:Methylibium (rho=0.53), the fungal OTU 37_4836_c:Sordariomycetes and the bacterial Otu00039_f:Cytophagaceae (rho=0.58) and finally between the 12_355_s:Tomentella ellisii and the bacterial Otu00015_c:TM7_1 (rho=0.64).

  literature. Some studies such as Mesa et al. (2017) studied the rhizospheric and endospheric bacterial communities associated with Betula celtiberica growing at a contaminated abandoned fertiliser industrial site in NW Spain. Data of the number of observed OTUs and Chao index found in roots of B. celtiberica by these authors are comparable with those found in the present study.

Mesa

  et al. (2017) observed in roots of B. celtiberica, that the phylum Proteobacteria was dominated by taxa related to Burkholderiales, and Pseudomonadales. However, in our study, the phylum Proteobacteria was mostly represented by the family Hyphomicrobiaceae (order Rhizobiales) and the genera Devosia and Rhodoplanes, whereas Pseudomonas, (which are frequently described in contaminated soils) were very rare (only three OTUs belonging to the Pseudomonas genera were observed (Otu00824,

Figure legends Figure 1

 legends1 Figure legends

Figure 2

 2 Figure 2 Composition of the root-associated bacterial (a) and fungal (b) communities.

Figure 3

 3 Figure 3 Heatmap and hierarchical cluster analysis of the most abundant bacterial and fungal OTUs (>0.5%). The dendrogram represents linkage clustering using Euclidean distance measures. OTU delineation was based on a threshold of <97% sequence similarity. Assignments between brackets show the lowest taxonomic level associated with the OTU: k kingdom, p phylum, o order, c class, f family, g genus, s genus_species

Figure 1

 1 Figure 1 Map of sampling points for each plant species. Betula pendula sampled at the center of the dump and named as B. pendula. Plants of P. tremula, P. nigra and S. purpurea were sampled at the edges. Plants of B. pendula growing in association with these species at the edges were also sampled and named as B. pendula-(Pt), B. pendula-(Pn) and B. pendula-(Sp). Only one soil sampling point was carried out for each plant pair.

Figure 2

 2 Figure 2 (a) Composition of the bacterial communities from the different roots at the family level and (b) composition of the fungal communities from the different roots at the family level. Only the 20 most abundant identified families are presented.

Table 4 .

 4 Indicator fungal and bacterial OTUs of B. pendula plants Associations were calculated with the Dufrene-Legendre indicator species analysis routine in R. Significance levels: *P ≤ 0.05; **P ≤ 0.01.

			stat	p.value	Phyllum	Class	Order	Family	Genus / Species
		Otu01003	0.775	0.001 ***	Proteobacteria	Gammaproteobacteria	unclass	unclass	unclass
		Otu00390	0.721	0.004 **	Bacteroidetes	Cytophagia	Cytophagales	unclass	unclass
		Otu00260	0.655	0.001 ***	Proteobacteria	Alphaproteobacteria	Rhizobiales	unclass	unclass
		Otu01224	0.636	0.006 **	Proteobacteria	Alphaproteobacteria	Rhizobiales	unclass	unclass
	BACTERIA	Otu00475 Otu00331 Otu00799 Otu00429 Otu00480 Otu00416	0.618 0.612 0.604 0.566 0.566 0.555	0.004 ** 0.005 ** 0.006 ** 0.01 ** 0.012 * 0.008 **	Proteobacteria Actinobacteria Proteobacteria Proteobacteria unclass Proteobacteria	unclass Actinobacteria Alphaproteobacteria Alphaproteobacteria unclass Alphaproteobacteria	unclass Actinomycetales Rhizobiales Rhizobiales unclass Rhizobiales	unclass Streptomycetaceae Hyphomicrobiaceae Hyphomicrobiaceae unclass unclass	unclass unclass Rhodoplanes unclass unclass unclass
		Otu00574	0.53	0.017 *	Proteobacteria	Alphaproteobacteria	unclass	unclass	unclass
		Otu01267	0.516	0.036 *	Proteobacteria	Alphaproteobacteria	unclass	unclass	unclass
		Otu00716	0.5	0.038 *	Actinobacteria	Actinobacteria	Actinomycetales	Micromonosporaceae	Catellatospora
		Otu01261	0.474	0.041 *	unclass	unclass	unclass	unclass	unclass
	FUNGI	3_5515 41_79123 1_99723 4_35502	0.993 0.735 0.577 0.572	0.001 *** 0.005 ** 0.002 ** 0.007 **	Ascomycota Basidiomycota Ascomycota Basidiomycota	Dothideomycetes Agaricomycetes Sordariomycetes Agaricomycetes	Incertae_sedis_8 Agaricales Hypocreales Russulales	Myxotrichaceae Unclass Unclass Russulaceae	Oidiodendron_maius|SH217755.06FU Unclass Unclass Russula

Figure 4

Non-parametric multidimensional scaling (NMDS) plot of fungal and bacterial abundant OTUs (>0.5%) communities associated with the roots of B. pendula, P. tremula, P. nigra and S. purpurea. Each point represents the bacterial community of a given root.

Each color represents one of the four trees. Log 10 abundance