Limited effects of crop foliar Si fertilization on a marginal soil under a future climate scenario.

Growing crops on marginal lands is a promising solution to alleviate the increasing pressure on agricultural land in Europe. Such crops will however be at the same time exposed to increased drought and pathogen prevalence, on already challenging soil conditions. Some sustainable practices, such as Silicon (Si) foliar fertilization, have been proposed to alleviate these two stress factors, but have not been tested under controlled, future climate conditions. We hypothesized that Si foliar fertilization would be beneficial for crops under future climate, and would have cascading beneficial effects on ecosystem processes, as many of them are directly dependent on plant health. We tested this hypothesis by exposing spring barley growing on marginal soil macrocosms (three with, three without Si treatment) to 2070 climate projections in an ecotron facility. Using the high-capacity monitoring of the ecotron, we estimated C, water, and N budgets of every macrocosm. Additionally, we measured crop yield, the biomass of each plant organ, and characterized bacterial communities using metabarcoding. Despite being exposed to water stress conditions, plants did not produce more biomass with the foliar Si fertilization, whatever the organ considered. Evapotranspiration (ET) was unaffected, as well as water quality and bacterial communities. However, in the 10-day period following two of the three Si applications, we measured a significant increase in C sequestration, when climate conditions where significantly drier, while ET remained the same. We interpreted these results as a less significant effect of Si treatment than expected as compared with literature, which could be explained by the high CO2 levels under future climate, that reduces need for stomata opening, and therefore sensitivity to drought. We conclude that making marginal soils climate proof using foliar Si treatments may not be a sufficient strategy, at least in this type of nutrient-poor, dry, sandy soil.

B-GATA factors are required to repress high-light stress responses in Marchantia polymorpha and Arabidopsis thaliana.

GATAs are evolutionarily conserved zinc-finger transcription factors from eukaryotes. In plants, GATAs can be subdivided into four classes, A-D, based on their DNA-binding domain, and into further subclasses based on additional protein motifs. B-GATAs with a so-called leucine-leucine-methionine (LLM)-domain can already be found in algae. In angiosperms, the B-GATA family is expanded and can be subdivided in to LLM- or HAN-domain B-GATAs. Both, the LLM- and the HAN-domain are conserved domains of unknown biochemical function. Interestingly, the B-GATA family in the liverwort Marchantia polymorpha and the moss Physcomitrium patens is restricted to one and four family members, respectively. And, in contrast to vascular plants, the bryophyte B-GATAs contain a HAN- as well as an LLM-domain. Here, we characterise mutants of the single B-GATA from Marchantia polymorpha. We reveal that this mutant has defects in thallus growth and in gemma formation. Transcriptomic studies uncover that the B-GATA mutant displays a constitutive high-light (HL) stress response, a phenotype that we then also confirm in mutants of Arabidopsis thaliana LLM-domain B-GATAs, suggesting that the B-GATAs have a protective role towards HL stress.

Resilience of barley (Hordeum vulgare) plants upon exposure to tramadol: Implication for the root-associated bacterial community and the antioxidative plant defence system.

Insufficiently treated reclaimed water can act as a source of contamination by introducing recalcitrant contaminants (e.g., pharmaceutical compounds) to various water bodies and/or agricultural soils after irrigation. Tramadol (TRD) is one of these pharmaceuticals that can be detected in influents and effluents of wastewater treatment plants, at discharge points as well as in surface waters in Europe. While the uptake of TRD by plants through irrigation water has been shown, plant responses towards this compound are still unclear. Therefore, this study aims to evaluate the effects of TRD on selected plant enzymes as well as on the root bacterial community structure. A hydroponic experiment was conducted to test the effects of TRD (100 µg L-1 TRD) on barley plants, at two harvesting time points after treatment. Accumulation of TRD in root tissues over time was observed reaching concentrations of 111.74 and 138.39 µg g-1 in total root FW after 12 and 24 days of exposure, respectively. Furthermore, noticeable inductions in guaiacol peroxidase (5.47-fold), catalase (1.83-fold) and glutathione S-transferase (3.23- and 2.09-fold) were recorded in roots of TRD-treated plants compared to controls after 24 days. A significant alteration in the beta diversity of root-associated bacteria due to TRD treatment was observed. Three amplicon sequence variants assigned to Hydrogenophaga, U. Xanthobacteraceae and Pseudacidovorax were differentially abundant in TRD-treated compared to control plants at both harvesting time points. This study reveals the resilience of plants through the induction of the antioxidative system and changes in the root-associated bacterial community to cope with the TRD metabolization/detoxification process.

Exudates from Miscanthus x giganteus change the response of a root-associated Pseudomonas putida strain towards heavy metals.

The composition of root exudates is modulated by several environmental factors, and it remains unclear how that affects beneficial rhizosphere or inoculated microorganisms under heavy metal (HM) contamination. Therefore, we evaluated the transcriptional response of Pseudomonas putida E36 (a Miscanthus x giganteus isolate with plant growth promotion-related properties) to Cd, Pb and Zn in an in vitro study implementing root exudates from M. x giganteus. To collect root exudates and analyse their composition plants were grown in a pot experiment under HM and control conditions. Our results indicated higher exudation rate for plants challenged with HM. Further, out of 29 organic acids identified and quantified in the root exudates, 8 of them were significantly influenced by HM (e.g., salicylic and terephthalic acid). The transcriptional response of P. putida E36 was significantly affected by the HM addition to the growth medium, increasing the expression of several efflux pumps and stress response-related functional units. The additional supplementation of the growth medium with root exudates from HM-challenged plants resulted in a downregulation of 29% of the functional units upregulated in P. putida E36 as a result of HM addition to the growth medium. Surprisingly, root exudates + HM downregulated the expression of P. putida E36 functional units related to plant colonization (e.g., chemotaxis, motility, biofilm formation) but upregulated its antibiotic and biocide resistance compared to the control treatment without HM. Our findings suggest that HM-induced changes in root exudation pattern may attract beneficial bacteria that are in turn awarded with organic nutrients, helping them cope with HM stress. However, it might affect the ability of these bacteria to colonize plants growing in HM polluted areas. Those findings may offer an insight for future in vivo studies contributing to improvements in phytoremediation measures.

Fate and impact of wastewater-borne micropollutants in lettuce and the root-associated bacteria.

The reuse of water for agricultural practices becomes progressively more important due to increasing demands for a transition to a circular economy. Treated wastewater can be an alternative option of blue water used for the irrigation of crops but its risks need to be evaluated. This study assesses the uptake and metabolization of pharmaceuticals and personal care products (PPCPs) derived from treated wastewater into lettuce as well as the impact on root-associated bacteria under a realistic and worst-case scenario. Lettuce was grown in a controlled greenhouse and irrigated with water or treated wastewater spiked with and without a mixture of fourteen different PPCPs at 10 µg/L or 100 µg/L. After harvesting the plants, the same soil was reused for a consecutive cultivation campaign to test for the accumulation of PPCPs. Twelve out of fourteen spiked PPCPs were detected in lettuce roots, and thirteen in leaves. In roots, highest concentrations were measured for sucralose, sulfamethoxazole and citalopram, while sucralose, acesulfame and carbamazepine were the highest in leaves. Higher PPCP concentrations were found in lettuce roots irrigated with spiked treated wastewater than in those irrigated with spiked water. The absolute bacterial abundance remained stable over both cultivation campaigns and was not affected by any of the treatments (type of irrigation water (water vs. wastewater) nor concentration of PPCPs). However, the irrigation of lettuce with treated wastewater had a significant effect on the microbial α-diversity indices at the end of the second cultivation campaign, and modified the structure and community composition of root-associated bacteria at the end of both campaigns. Five and fourteen bacterial families were shown to be responsible for the observed changes at the end of the first and second cultivation campaign, respectively. Relative abundance of Haliangium and the clade Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium was significantly affected in response to PCPPs exposure. Caulobacter, Cellvibrio, Hydrogenophaga and Rhizobacter were significantly affected in microcosms irrigated with wastewater.

Plant GATA Factors: Their Biology, Phylogeny, and Phylogenomics.

GATA factors are evolutionarily conserved transcription factors that are found in animals, fungi, and plants. Compared to that of animals, the size of the plant GATA family is increased. In angiosperms, four main GATA classes and seven structural subfamilies can be defined. In recent years, knowledge about the biological role and regulation of plant GATAs has substantially improved. Individual family members have been implicated in the regulation of photomorphogenic growth, chlorophyll biosynthesis, chloroplast development, photosynthesis, and stomata formation, as well as root, leaf, and flower development. In this review, we summarize the current knowledge of plant GATA factors. Using phylogenomic analysis, we trace the evolutionary origin of the GATA classes in the green lineage and examine their relationship to animal and fungal GATAs. Finally, we speculate about a possible conservation of GATA-regulated functions across the animal, fungal, and plant kingdoms.

Removal of tramadol from water using Typha angustifolia and Hordeum vulgare as biological models: Possible interaction with other pollutants in short-term uptake experiments.

Tramadol (TRD) is widely detected in aquatic ecosystems as a result of massive abuse and insufficient removal from wastewater facilities. As a result, TRD can contaminate groundwater sources and/or agricultural soils. While TRD toxicity has been reported from aquatic biota, data about TRD detection in plants are scarce. Moreover, information regarding plant capability for TRD removal is lacking. To understand the fate of this opioid, we have investigated the uptake, translocation and removal capacity of TRD by plants, addressing short-term and long-term uptake. The uptake rates of TRD, in excised barley and cattail roots, were 5.18 and 5.79 µg g-1 root fresh weight day-1, respectively. However, TRD uptake was strongly inhibited after co-exposing these roots either with the drug venlafaxine (similar molecular structure as TRD) or with quinidine (an inhibitor of cellular organic cation transporters). When barley seedlings were exposed to TRD in a hydroponic experiment a removal efficiency up to 90% (within 15 days) was obtained, with bioconcentration and translocation factors close to 9 and 1, respectively. The combination of results from both plants and the inhibition observed after treatment with quinidine revealed that organic cation transporters may be involved in the uptake of TRD by plants.

The changes in Lemna minor metabolomic profile: A response to diclofenac incubation.

Metabolomics is an emerging approach that investigates the changes in the metabolome profile. In the present study, Lemna minor -considered as an experimental aquatic plant model- was incubated with 10 and 100 µM diclofenac (DCF) for 96 h, respectively. Knowing that DCF is internationally often problematic in wastewater effluents and that it might affect particularly the metabolic profiles in aquatic plants, mainly the oxidoreductase, dehydrogenase, peroxidase, and glutathione reductase activities, here it was hypothesized (H) that in the common duckweed, DCF might increase the phenolic and flavonoids pathways, as an antioxidant response to this stress (H1). Also, it was expected DCF to alternate the physiological characteristics, especially the molecular interaction and biochemical properties, of Lemna (H2). Metabolic changes were investigated with target and untargeted screening analysis using RPLC-HILIC-ESI-TOF-MS. Twelve amino acids were identified in all treatments, together with three organic acids (p-coumaric, cinnamic, and sinapic acids). In untargeted screening, the important metabolites to discriminate between different treatments were assigned to Lemna such as organic acids, lignin, sugars, amino acids, dipeptides, flavonoids, biflavonoids, fatty acids, among others. In resume, Lemna responded to both DCF concentrations, showing different stress patterns. A similar metabolic response had already been identified in other studies in exposing Lemna to other anthropogenic stressors (like pesticides).

Relaunch cropping on marginal soils by incorporating amendments and beneficial trace elements in an interdisciplinary approach.

In the EU and world-wide, agriculture is in transition. Whilst we just converted conventional farming imprinted by the post-war food demand and heavy agrochemical usage into integrated and sustainable farming with optimized production, we now have to focus on even smarter agricultural management. Enhanced nutrient efficiency and resistance to pests/pathogens combined with a greener footprint will be crucial for future sustainable farming and its wider environment. Future land use must embrace efficient production and utilization of biomass for improved economic, environmental, and social outcomes, as subsumed under the EU Green Deal, including also sites that have so far been considered as marginal and excluded from production. Another frontier is to supply high-quality food and feed to increase the nutrient density of staple crops. In diets of over two-thirds of the world's population, more than one micronutrient (Fe, Zn, I or Se) is lacking. To improve nutritious values of crops, it will be necessary to combine integrated, systems-based approaches of land management with sustainable redevelopment of agriculture, including central ecosystem services, on so far neglected sites: neglected grassland, set aside land, and marginal lands, paying attention to their connectivity with natural areas. Here we need new integrative approaches which allow the application of different instruments to provide us not only with biomass of sufficient quality and quantity in a site specific manner, but also to improve soil ecological services, e.g. soil C sequestration, water quality, habitat and soil resistance to erosion, while keeping fertilization as low as possible. Such instruments may include the application of different forms of high carbon amendments, the application of macro- and microelements to improve crop performance and quality as well as a targeted manipulation of the soil microbiome. Under certain caveats, the potential of such sites can be unlocked by innovative production systems, ready for the sustainable production of crops enriched in micronutrients and providing services within a circular economy.

Untargeted Analysis of Lemna minor Metabolites: Workflow and Prioritization Strategy Comparing Highly Confident Features between Different Mass Spectrometers.

Metabolomics approaches provide a vast array of analytical datasets, which require a comprehensive analytical, statistical, and biochemical workflow to reveal changes in metabolic profiles. The biological interpretation of mass spectrometric metabolomics results is still obstructed by the reliable identification of the metabolites as well as annotation and/or classification. In this work, the whole Lemna minor (common duckweed) was extracted using various solvents and analyzed utilizing polarity-extended liquid chromatography (reversed-phase liquid chromatography (RPLC)-hydrophilic interaction liquid chromatography (HILIC)) connected to two time-of-flight (TOF) mass spectrometer types, individually. This study (introduces and) discusses three relevant topics for the untargeted workflow: (1) A comparison study of metabolome samples was performed with an untargeted data handling workflow in two different labs with two different mass spectrometers using the same plant material type. (2) A statistical procedure was observed prioritizing significant detected features (dependent and independent of the mass spectrometer using the predictive methodology Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA). (3) Relevant features were transferred to a prioritization tool (the FOR-IDENT platform (FI)) and were compared with the implemented compound database PLANT-IDENT (PI). This compound database is filled with relevant compounds of the Lemnaceae, Poaceae, Brassicaceae, and Nymphaceae families according to analytical criteria such as retention time (polarity and LogD (pH 7)) and accurate mass (empirical formula). Thus, an untargeted analysis was performed using the new tool as a prioritization and identification source for a hidden-target screening strategy. Consequently, forty-two compounds (amino acids, vitamins, flavonoids) could be recognized and subsequently validated in Lemna metabolic profile using reference standards. The class of flavonoids includes free aglycons and their glycosides. Further, according to our knowledge, the validated flavonoids robinetin and norwogonin were for the first time identified in the Lemna minor extracts.

Cell Walls Are Remodeled to Alleviate nY2O3 Cytotoxicity by Elaborate Regulation of de Novo Synthesis and Vesicular Transport.

Yttrium oxide nanoparticles (nY2O3), one of the broadly used rare earth nanoparticles, can interact with plants and possibly cause plant health and environmental impacts, but the plant defense response particularly at the nanoparticle-cell interface is largely unknown. To elucidate this, Bright Yellow 2 (BY-2) tobacco (Nicotiana tabacum L.) suspension-cultured cells were exposed to 50 mg L-1 nY2O3 (30 nm) for 12 h. Although 42.2% of the nY2O3 remained outside of protoplasts, nY2O3 could still traverse the cell wall and was partially deposited inside the vacuole. In addition to growth inhibition, morphological and compositional changes in cell walls occurred. Together with a locally thickened (7-13-fold) cell wall, increased content (up to 58%) of pectin and reduction in (up to 29%) hemicellulose were observed. Transcriptome analysis revealed that genes involved in cell wall metabolism and remodeling were highly regulated in response to nY2O3 stress. Expression of genes for pectin synthesis and degradation was up- and down-regulated by 31-78% and 13-42%, respectively, and genes for xyloglucan and pectin modifications were up- and down-regulated by 82% and 81-92%, respectively. Interestingly, vesicle trafficking seemed to be activated, enabling the repair and defense against nY2O3 disturbance. Our findings indicate that, although nY2O3 generated toxicity on BY-2 cells, it is very likely that during the recovery process cell wall remodeling was initiated to gain resistance to nY2O3 stress, demonstrating the plant's cellular regulatory machinery regarding repair and adaptation to nanoparticles like nY2O3.

Post-reclamation microbial diversity and functions in hexachlorocyclohexane (HCH) contaminated soil in relation to spontaneous HCH tolerant vegetation.

The toxicity, volatility and persistence of the obsolete organochlorine pesticide hexachlorocyclohexane (HCH), makes reclamation of contaminated areas a priority for the health and welfare of neighboring human communities. Microbial diversity and functions and their relation to spontaneous vegetation in post-excavation situations, are essential indicators to consider in bioaugmentation or microbe-assisted phytoremediation strategies at field scale. Our study aimed to evaluate the effects of long-term HCH contamination on soil and plant-associated microbial communities, and whether contaminated soil has the potential to act as a bacterial inoculum in post-excavation bioremediation strategies. To scrutinize the role of vegetation, the potential nitrogen fixation of free-living and symbiotic diazotrophs of the legume Lotus tenuis was assessed as a measure of nutrient cycling functions in soil under HCH contamination. Potential nitrogen fixation was generally not affected by HCH, with the exception of lower nifH gene counts in excavated contaminated rhizospheres, most probably a short-term HCH effect on early bacterial succession in this compartment. HCH shaped microbial communities in long-term contaminated bulk soil, where we identified possible HCH tolerants such as Sphingomonas and Altererythrobacter. In L. tenuis rhizosphere, microbial community composition was additionally influenced by plant growth stage. Sphingobium and Massilia were the bacterial genera characteristic for HCH contaminated rhizospheres. Long-term HCH contamination negatively affected L. tenuis growth and development. However, root-associated bacterial community composition was driven solely by plant age, with negligible HCH effect. Results showed that L. tenuis acquired possible HCH tolerant bacteria such as the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade, Sphingomonas, Massilia or Pantoea which could simultaneously offer plant growth promoting (PGP) benefits for the host. Finally, we identified an inoculum with possibly HCH tolerant, PGP bacteria transferred from the contaminated bulk soil to L. tenuis roots through the rhizosphere compartment, consisting of Mesorhizobium loti, Neorhizobium galegae, Novosphingobium lindaniclasticum, Pantoea agglomerans and Lysobacter bugurensis.

Effect of the pharmaceuticals diclofenac and lamotrigine on stress responses and stress gene expression in lettuce (Lactuca sativa) at environmentally relevant concentrations.

Vegetable crops irrigated with treated wastewater can take up the environmentally persistent pharmaceuticals diclofenac and lamotrigine. This study aimed at quantifying the uptake and translocation of the two pharmaceuticals in lettuce (Lactuca sativa) as well as on the elucidation of the molecular and physiological changes triggered by them. Therefore, plants were cultivated in a phytochamber in hydroponic systems under controlled conditions and treated independently with diclofenac (20 µg L-1) and lamotrigine (60 µg L-1) for 48 h. A low translocation of lamotrigine but not of diclofenac or its metabolite 4'-hydroxydiclofenac to leaves was observed, which corresponded with the expression of stress related genes only in roots of diclofenac treated plants. We observed an oxidative burst in roots and leaves occurring around the same time point when lamotrigine was detected in leaves. This could be responsible for the significantly changed gene expression pattern in both tissues. Our results showed for the first time that pharmaceuticals like lamotrigine or diclofenac might act as signals or zeitgebers, affecting the circadian expression of stress related genes in lettuce possibly causing a repressed physiological status of the plant.

Untargeted Metabolomics Studies on Drug-Incubated Phragmites australis Profiles.

Plants produce a huge number of functionally and chemically different natural products that play an important role in linking the plant with the adjacent environment. Plants can also absorb and transform external organic compounds (xenobiotics). Currently there are only a few studies concerning the effects of xenobiotics and their transformation products on plant metabolites using a mass spectrometric untargeted screening strategy. This study was designed to investigate the changes of the Phragmites australis metabolome following/after diclofenac or carbamazepine incubation, using a serial coupling of reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) combined with accurate high-resolution time-of-flight mass spectrometer (TOF-MS). An untargeted screening strategy of metabolic fingerprints was developed to purposefully compare samples from differently treated P. australis plants, revealing that P. australis responded to each drug differently. When solvents with significantly different polarities were used, the metabolic profiles of P. australis were found to change significantly. For instance, the production of polyphenols (such as quercetin) in the plant increased after diclofenac incubation. Moreover, the pathway of unsaturated organic acids became more prominent, eventually as a reaction to protect the cells against reactive oxygen species (ROS). Hence, P. australis exhibited an adaptive mechanism to cope with each drug. Consequently, the untargeted screening approach is essential for understanding the complex response of plants to xenobiotics.

Response of Barley Plants to Drought Might Be Associated with the Recruiting of Soil-Borne Endophytes.

Mechanisms used by plants to respond to water limitation have been extensively studied. However, even though the inoculation of beneficial microbes has been shown to improve plant performance under drought stress, the inherent role of soil microbes on plant response has been less considered. In the present work, we assessed the importance of the soil microbiome for the growth of barley plants under drought stress. Plant growth was not significantly affected by the disturbance of the soil microbiome under regular watering. However, after drought stress, we observed a significant reduction in plant biomass, particularly of the root system. Plants grown in the soil with disturbed microbiome were significantly more affected by drought and did not recover two weeks after re-watering. These effects were accompanied by changes in the composition of endophytic fungal and bacterial communities. Under natural conditions, soil-derived plant endophytes were major colonizers of plant roots, such as Glycomyces and Fusarium, whereas, for plants grown in the soil with disturbed microbiome seed-born bacterial endophytes, e.g., Pantoea, Erwinia, and unclassified Pseudomonaceae and fungal genera normally associated with pathogenesis, such as Gibberella and Gaeumannomyces were observed. Therefore, the role of the composition of the indigenous soil microbiota should be considered in future approaches to develop management strategies to make plants more resistant towards abiotic stress, such as drought.

52 years of ecological restoration following a major disturbance by opencast lignite mining does not reassemble microbiome structures of the original arable soils.

Opencast mining for lignite continuously creates areas of land that require restoration. Here we applied a chronosequence approach to investigate the development of soil bacterial communities during 52 years as influenced by the restoration process and subsequent changes in soil physico-chemical conditions starting from the initial reclamation of the sites. By comparison with the unaffected soils near the mine, we were able to address the question if soil bacterial communities have reached a steady state within 52 years, which is comparable to the original soil. Our study revealed three distinct phases of the restoration process, each with a specific bacterial community composition. The effect size of these changes was similar to the one observed for seasonal dynamics at our sites. At the beginning of the restoration process Flavobacteriaceae, Cytophagaceae and Sphingobacteriaceae were found as typical members of the bacterial community as well as Rhizobiales as a result of the cultivation of alfalfa on the restored plots. At later stage the families Peptostreptococcaceae, Desulfurellaceae as well as Streptomycetaceae increased in relative abundance and became dominant members of the bacterial community. Even though overall bacterial abundance and richness exhibited values comparable to the original soil already 5 years after the start of the restoration process, main responder analyses reveal differences in the bacterial community structure even 52 years after the start of the restoration process. Mostly Nitrospirae were reduced in abundance in the soils restored for 52 years compared to the original soils. To broaden the significance of our study, we compared our data bioinformatically with published results from other restored areas, which were previously affected by opencast mining. Despite different durations of the different restoration phase, we could observe a large degree of conformity when bacterial patterns of succession were compared indicating common modes of action of ecological restoration tools for bacterial communities.

Lemna minor studies under various storage periods using extended-polarity extraction and metabolite non-target screening analysis.

Plant metabolomic studies cover a broad band of compounds, including various functional groups with different polarities and other physiochemical properties. For this reason, specific optimized methods are needed in order to enable efficient and non-destructive extraction of molecules over a large range of LogD values. This study presents a simple and efficient extraction procedure for Lemna minor samples demonstrating polarity extension of the molecular range. The Lemna samples chosen were kept under the following storage conditions: 1) fresh, 2) stored for a few days at -80 °C, and 3) stored for 6 months at -80 °C. The samples were extracted using five specifically chosen solvents: 100 % ethanol, 100 % methanol (MeOH), acidic 90 % MeOH (MeOH-water-formic acid (FAC) (90:9.5:0.5, v/v/v), MeOH-water (50:50, v/v), and 100 % water. The final extraction procedure was conducted subject to three solvent conditions, and the subsequent polarity-extended analysis was applied for Lemna minor samples using RPLC-HILIC-ESI-TOF-MS. The extraction yield is in descending order (acidic 90 % MeOH), 50 % MeOH, 100 % water and 100 % MeOH. The results displayed significant molecular differences, both in the extracts investigated and in the fresh Lemna samples, compared to stored samples, in terms of the extraction yield and reducing contents as well as the number of features. The storage of Lemna minor resulted in changes to the fingerprint of its metabolites as the reducing contents increased. The comparisons enable a direct view of molecule characterizations, in terms of their polarity, molecular mass, and signal intensity. This parametric information would appear ideal for further statistical data analysis. Consequently, the extraction procedure and the analysis/data evaluation are highly suitable for the so-called extended-polarity non-target screening procedure.

Enrichment of endophytic Actinobacteria in roots and rhizomes of Miscanthus × giganteus plants exposed to diclofenac and sulfamethoxazole.

This study investigates how wastewater containing 2 mg l-1 of sulfamethoxazole (SMX) and 2 mg l-1 of diclofenac (DCF) affects the composition of bacterial communities present in the roots and rhizomes of Miscanthus × giganteus plants grown in laboratory-scale constructed wetlands. Bacterial communities in plant roots and rhizomes were identified in treated and control samples by 16S rRNA amplicon sequencing. Moreover, bacterial endophytes were isolated in R2A and 1/10 869 media and screened for their ability to metabolize SMX and DCF in liquid medium by HPLC. Our results show significant changes in the abundance of main genera, namely Sphingobium and Streptomyces between control and treated plants. Around 70% of the strains isolated from exposed plants belonged to the phylum Actinobacteria and were classified as Streptomyces, Microbacterium, and Glycomyces. In non-exposed plants, Proteobacteria represented 43.5% to 63.6% of the total. We identified 17 strains able to remove SMX and DCF in vitro. From those, 76% were isolated from exposed plants. Classified mainly as Streptomyces, they showed the highest SMX (33%) and DCF (41%) removal efficiency. These isolates, alone or in combination, might be used as bio-inoculants in constructed wetlands to enhance the phytoremediation of SMX and DCF during wastewater treatment.

Changes induced by heavy metals in the plant-associated microbiome of Miscanthus x giganteus.

Miscanthus x giganteus is a high biomass producing plant with tolerance to heavy metals. This makes Miscanthus interesting to be used for phytoremediation of heavy metal contaminated areas coupled with energy production. Since plant performance in metal polluted areas is impaired, their growth and phytoremediation effect can be improved with bacterial assistance. To identify positive and negative responders of M. x giganteus associated microbiome influenced by Cd, Pb and Zn stress compared to non-contaminated controls, we designed a greenhouse experiment. Structure of the bacterial community in three rhizocompartments, namely rhizosphere, rhizoplane and root endosphere was analysed using an isolation independent molecular approach based on 16S rRNA gene barcoding. Furthermore, quantitative PCR (qPCR) was used for bacterial biomass estimation. Our results indicated that biomass and total bacterial diversity in rhizosphere, rhizoplane and root endosphere did not significantly change despite of substantial root uptake of heavy metals. Overall, we detected 6621 OTUs, from which 171 were affected by metal addition. Whereas Streptomyces and Amycolatopsis taxa were negatively affected by the heavy metal treatment in endosphere, taxa assigned to Luteolibacter in rhizosphere and rhizoplane (log2 fold change 1.9-4.1) and Micromonospora in endosphere (log2 fold change 10.2) were found to be significantly enriched and highly abundant (0.1-3.7% relative abundance) under heavy metal stress. Those taxa might be of key importance for M. x giganteus performance under heavy metal pollution and might be interesting candidates for the development of new bioinocula in the future to promote plant growth and phytoremediation in heavy metal contaminated soils.

Under temperate climate, the conversion of grassland to arable land affects soil nutrient stocks and bacteria in a short term.

Projected population growth and climate change will make it inevitable to convert neglected and marginal land into productive arable land. We investigate the influence of agricultural management practices on nutrient stocks and soil functions during the conversion of former extensively used grassland to arable land. Effects of grassland removal, tillage, intercropping with faba bean (Vicia faba) and its later incorporation were studied with respect to soil properties and bacterial community structure. Therefore, composite samples were collected with a core sampler from the topsoil (0-20 cm) in (a) the initial grassland, (b) the transitional phase during the vegetation period of V. faba, (c) after ploughing the legume in, and (d) untreated controls. In all samples, nitrate-N, ammonium-N, dissolved organic carbon (DOC) and total nitrogen bound (TNb) were analyzed and comparisons of the bacterial community structure after 16S-amplicon sequencing were performed to assess soil functions. Mineralization after grassland conversion followed by the biological nitrogen fixation of broad beans enhanced the nitrate-N content in bulk soil from 4 to almost 50 µg N g-1dw. Bacterial community structure on phylum level in bulk soil was dominated by Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, and Bacteroidetes and remained almost stable. However, alpha and beta-diversity analysis revealed a change of the bacterial composition at the final state of the conversion. This change was primarily driven by increasing abundances of the genera Massilia and Lysobacter, both members of the Proteobacteria, after the decay of the leguminous plant residues. Furthermore, increasing abundances of the family Gaiellaceae and its genus Gaiella fostered this change and were related to the decreasing carbon to nitrogen ratio. In short, gentle management strategies could replace the input of mineral fertilizer with the aim to contribute to future sustainable and intensified production even on converted grassland.