Rhizobium symbiosis improves amino acid and secondary metabolite biosynthesis of tungsten-stressed soybean (Glycine max).

The industrially important transition metal tungsten (W) shares certain chemical properties with the essential plant micronutrient molybdenum and inhibits the activity of molybdoenzymes such as nitrate reductase, impacting plant growth. Furthermore, tungsten appears to interfere with metabolic processes on a much wider scale and to trigger common heavy metal stress response mechanisms. We have previously found evidence that the tungsten stress response of soybeans (Glycine max) grown with symbiotically associated N2-fixing rhizobia (Bradyrhizobium japonicum) differs from that observed in nitrogen-fertilized soy plants. This study aimed to investigate how association with symbiotic rhizobia affects the primary and secondary metabolite profiles of tungsten-stressed soybean and whether changes in metabolite composition enhance the plant's resilience to tungsten. This comprehensive metabolomic and proteomic study presents further evidence that the tungsten-stress response of soybean plants is shaped by associated rhizobia. Symbiotically grown plants (N fix) were able to significantly increase the synthesis of an array of protective compounds such as phenols, polyamines, gluconic acid, and amino acids such as proline. This resulted in a higher antioxidant capacity, reduced root-to-shoot translocation of tungsten, and, potentially, also enhanced resilience of N fix plants compared to non-symbiotic counterparts (N fed). Taken together, our study revealed a symbiosis-specific metabolic readjustment in tungsten-stressed soybean plants and contributed to a deeper understanding of the mechanisms involved in the rhizobium-induced systemic resistance in response to heavy metals.

Phytosiderophore pathway response in barley exposed to iron, zinc or copper starvation.

Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption. Enhanced exudation and re-uptake of metal chelators, so-called phytosiderophores, by roots of graminaceous plants has been implicated in efficient micronutrient acquisition. We compared PS biosynthesis and exudation as a response mechanism to either Fe, Zn or Cu starvation. Two barley (Hordeum vulgare L.) lines with contrasting micronutrient grain yields were grown hydroponically and PS exudation (LC-MS) and root gene expression (RNAseq) were determined after either Fe, Zn, or Cu starvation. The response strength of the PS pathway was micronutrient dependent and decreased in the order Fe > Zn > Cu deficiency. We observed a stronger expression of PS pathway genes and greater PS exudation in the barley line with large micronutrient grain yield suggesting that a highly expressed PS pathway might be an important trait involved in high micronutrient accumulation. In addition to several metal specific transporters, we also found that the expression of IRO2 and bHLH156 transcription factors was not only induced under Fe but also under Zn and Cu deficiency. Our study delivers important insights into the role of the PS pathway in the acquisition of different micronutrients.

Maize (Zea mays L.) root exudation profiles change in quality and quantity during plant development - A field study.

Deciphering root exudate composition of soil-grown plants is considered a crucial step to better understand plant-soil-microbe interactions affecting plant growth performance. In this study, two genotypes of Zea mays L. (WT, rth3) differing in root hair elongation were grown in the field in two substrates (sand, loam) in custom-made, perforated columns inserted into the field plots. Root exudates were collected at different plant developmental stages (BBCH 14, 19, 59, 83) using a soil-hydroponic-hybrid exudation sampling approach. Exudates were characterized by LC-MS based non-targeted metabolomics, as well as by photometric assays targeting total dissolved organic carbon, soluble carbohydrates, proteins, amino acids, and phenolics. Results showed that plant developmental stage was the main driver shaping both the composition and quantity of exuded compounds. Carbon (C) exudation per plant increased with increasing biomass production over time, while C exudation rate per cm² root surface area h-1 decreased with plant maturity. Furthermore, exudation rates were higher in the substrate with lower nutrient mobility (i.e., loam). Surprisingly, we observed higher exudation rates in the root hairless rth3 mutant compared to the root hair-forming WT sibling, though exudate metabolite composition remained similar. Our results highlight the impact of plant developmental stage on the plant-soil-microbe interplay.

Total synthesis of [13 C2 ]-labeled phytosiderophores of the mugineic and avenic acid families.

We, herein, report the synthesis of 13 C2 -labeled natural products from the mugineic acid and avenic acid family. These phytosiderophores ("plant iron carriers") are built up from non-proteinogenic amino acids and play a key role in micronutrient uptake in gramineous plants. In this work, two central building blocks are prepared from labeled starting materials (13 C2 -bromoacetic acid, 13 C2 -glycine) and further employed in our recently reported divergent, branched synthetic strategy delivering eight isotopically labeled phytosiderophores. The required labeled building blocks (13 C2 -l-allylglycine and a related hydroxylated derivative) were prepared via enantioselective phase-transfer catalysis and enantio- and diastereoselective aldol condensation with a chiral auxiliary, respectively, both potentially valuable themselves for other synthetic routes toward labeled (natural) products.

Surveying the mugineic acid family: Ion mobility - quadrupole time-of-flight mass spectrometry (IM-QTOFMS) characterization and tandem mass spectrometry (LC-ESI-MS/MS) quantification of all eight naturally occurring phytosiderophores.

Phytosiderophores (PS) are root exudates released by grass species (Poaceae) that play a pivotal role in iron (Fe) plant nutrition. A direct determination of PS in biological samples is of paramount importance in understanding micronutrient acquisition mediated by PS. To date, eight plant-born PS have been identified; however, no analytical procedure is currently available to quantify all eight PS simultaneously with high analytical confidence. With access to the full set of PS standards for the first time, we report comprehensive methods to both fully characterize (IM-QTOFMS) and quantify (LC-ESI-MS/MS) all eight naturally occurring PS belonging to the mugineic acid family. The quantitative method was fully validated, yielding linear results for all eight analytes, and no unwanted interferences with soil and plant matrices were observed. LOD and LOQ values determined for each PS were below 11 and 35 nmol L-1, respectively. The method's precision under reproducibility conditions (intra- and inter-day) of measurement was less than 2.5% RSD for all analytes. Additionally, all PS were annotated with high-resolution mass spectrometric fragment spectra and further characterized via drift tube ion mobility-mass spectrometry. The collision cross-sections obtained for primary ion species yielded a valuable database for future research focused on in-depth PS studies. The new quantitative method was applied to analyse root exudates from Fe-controlled and deficient barley, oat, rye, and sorghum plants. All eight PS, including mugineic acid (MA), 3"-hydroxymugineic acid (HMA), 3"-epi-hydroxymugineic acid (epi-HMA), hydroxyavenic acid (HAVA), deoxymugineic acid (DMA), 3"-hydroxydeoxymugineic acid (HDMA), 3"-epi-hydroxydeoxymugineic acid (epi-HDMA) and avenic acid (AVA) were for the first time successfully identified and quantified in root exudates of various graminaceous plants using a single analytical procedure. These newly developed methods can be applied to studies aimed at improving crop yield and micronutrient grain content for food consumption via plant-based biofortification.

Chemical imaging reveals environmental risk of minor tungsten and lead shotgun pellet constituents during weathering in soil.

Tungsten (W)-based shots are considered more environmentally safe than lead (Pb)-based shots, but knowledge about the W-shot fate in the soil environment is still limited, especially in terms of minor constituents such as iron, copper, and nickel (Ni). Contaminant behaviour in soil strongly depends on pH; in turn, the corrosion of metal composites may affect the pH locally. The aim of this study was to compare Pb- and W-shot weathering dynamics in soil (silt loam, pH 6.3) and reveal the interplay of shot weathering-induced pH-changes on the mobility of elements using in situ chemical imaging (Diffusive gradients in thin films for labile elements, planar optodes for soil pH) and batch incubation experiments over time (16 months). Despite our expectation to find acidification due to W oxidation, we observed a pH increase by 0.2 units in extracted soil solutions and by 0.6 units in the soil around W-shots as Ni dissolved from the binder phase of the shot. After 10 weeks, release of labile Ni was 3-times higher compared to W despite the low Ni content in the shot (7 %, m/m). Pb-shot oxidation increased soil solution pH by 0.5 units which likely supported mobility of Pb-shot-derived antimony (Sb). Steep gradients of labile W and Pb and soil solution concentrations <0.8 µmol L-1 indicated that transfer from shot to soil was low. Contrastingly, labile Ni and Sb were found up to ~4 mm from the shot surface and in higher soil solution concentrations as suggested by the shot constitution, indicating higher mobility of minor as compared to major shot constituents. After 16 months, 36 % of total Ni were dissolved in the soil solution highlighting the environmental relevance of minor shot constituents in Pb-shot alternatives after short term weathering in soil.

The effect of root hairs on exudate composition: a comparative non-targeted metabolomics approach.

Root exudation is a major pathway of organic carbon input into soils. It affects soil physical properties, element solubility as well as speciation, and impacts the microbial community in the rhizosphere. Root exudates contain a large number of primary and secondary plant metabolites, and the amount and composition are highly variable depending on plant species and developmental stage. Detailed information about exudate composition will allow for a better understanding of exudate-driven rhizosphere processes and their feedback loops. Although non-targeted metabolomics by high-resolution mass spectrometry is an established tool to characterize root exudate composition, the extent and depth of the information obtained depends strongly on the analytical approach applied. Here, two genotypes of Zea mays L., differing in root hair development, were used to compare six mass spectrometric approaches for the analysis of root exudates. Reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry (LC-TOF-MS), as well as direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS), were applied with positive and negative ionization mode. By using the same statistical workflow, the six approaches resulted in different numbers of detected molecular features, ranging from 176 to 889, with a fraction of 48 to 69% of significant features (fold change between the two genotypes of > 2 and p-value < 0.05). All approaches revealed the same trend between genotypes, namely up-regulation of most metabolites in the root hair defective mutant (rth3). These results were in agreement with the higher total carbon and nitrogen exudation rate of the rth3-mutant as compared to the corresponding wild-type maize (WT). However, only a small fraction of features were commonly found across the different analytical approaches (20-79 features, 13-31% of the rth3-mutant up-regulated molecular formulas), highlighting the need for different mass spectrometric approaches to obtain a more comprehensive view into the composition of root exudates. In summary, 111 rth3-mutant up-regulated compounds (92 different molecular formulas) were detected with at least two different analytical approaches, while no WT up-regulated compound was found by both, LC-TOF-MS and DI-FT-ICR-MS. Zea mays L. exudate features obtained with multiple analytical approaches in our study were matched against the metabolome database of Zea mays L. (KEGG) and revealed 49 putative metabolites based on their molecular formula.

A quick and simple spectrophotometric method to determine total carbon concentrations in root exudate samples of grass species.

Purpose: Root exudates are key components driving belowground interaction between plant, microbes and soil. High-end analytical approaches provide advanced insights into exudate metabolite diversity, however, the amount of total carbon (C) released by roots should always be determined as the most basic parameter when characterizing root exudation as it (i) provides quantitative information of C exuded into the surrounding soil and (ii) allows to relate the abundance of individual exudate compounds to total C released. Here we propose a simple and quick, spectrophotometry-based method to quantify total dissolved organic carbon (DOC) concentration in exudation samples that is based on measuring the absorption of a pre-filtered but otherwise untreated exudate sample at 260 nm (DOC260). Method: Exudate samples collected from different grass genotypes (Zea mays, Oryza sativa, Hordeum vulgare) grown in various experimental settings (soil, hydroponic) were analysed with the DOC260 assay and results were compared with C concentrations obtained by liquid TOC-analyser. Conclusion: We demonstrated that the DOC260 method allowed for quick and inexpensive measurements of total dissolved organic carbon concentrations in exudate samples from grass species grown under nutrient sufficient as well as under P deficient conditions. Interestingly, DOC260 failed to predict DOC concentrations in exudate samples from plants grown under Zn and Fe deficiency suggesting a strong shift in metabolite composition under micronutrient deficiency. Even though the applicability of the DOC260 method remains to be tested on exudate samples originating from dicots and plants exposed to other environmental stresses (e.g. pathogen attack, heavy metal stress, etc), it will help to increase our understanding of root exudation and related rhizosphere processes in the future.

Harnessing belowground processes for sustainable intensification of agricultural systems.

Increasing food demand coupled with climate change pose a great challenge to agricultural systems. In this review we summarize recent advances in our knowledge of how plants, together with their associated microbiota, shape rhizosphere processes. We address (molecular) mechanisms operating at the plant-microbe-soil interface and aim to link this knowledge with actual and potential avenues for intensifying agricultural systems, while at the same time reducing irrigation water, fertilizer inputs and pesticide use. Combining in-depth knowledge about above and belowground plant traits will not only significantly advance our mechanistic understanding of involved processes but also allow for more informed decisions regarding agricultural practices and plant breeding. Including belowground plant-soil-microbe interactions in our breeding efforts will help to select crops resilient to abiotic and biotic environmental stresses and ultimately enable us to produce sufficient food in a more sustainable agriculture in the upcoming decades.

The breakthrough paradox: How focusing on one form of innovation jeopardizes the advancement of science: How focusing on one form of innovation jeopardizes the advancement of science.

Research needs a balance of risk-taking in "breakthrough projects" and gradual progress. For building a sustainable knowledge base, it is indispensable to provide support for both.

A Unified Approach to Phytosiderophore Natural Products.

This work reports on the concise total synthesis of eight natural products of the mugineic acid and avenic acid families (phytosiderophores). An innovative "east-to-west" assembly of the trimeric products resulted in a high degree of divergence enabling the formation of the final products in just 10 or 11 steps each with a minimum of overall synthetic effort. Chiral pool starting materials (l-malic acid, threonines) were employed for the outer building blocks while the middle building blocks were accessed by diastereo- and enantioselective methods. A highlight of this work consists in the straightforward preparation of epimeric hydroxyazetidine amino acids, useful building blocks on their own, enabling the first synthesis of 3''-hydroxymugineic acid and 3''-hydroxy-2'-deoxymugineic acid.

Response of tungsten (W) solubility and chemical fractionation to changes in soil pH and soil aging.

A thorough understanding of the geochemical behavior of W in soils is crucial for environmental risk assessment. Soil pH is known as master variable of element solubility and bioavailability in soils. Here we report on effects of soil pH (modified by liming and acid - base additions) and soil aging on the environmental availability of W in soil using W solubility and chemical fractionation as indicators. Experimental soils included two naturally acidic soils with contrasting soil texture (SAND, CLAY), at native pH or limed with 2.5% CaCO3, and spiked with increasing concentration of W. Our results showed that W was significantly more labile in alkaline compared to acidic soils, confirming the validity of results of pure-mineral studies for more heterogeneously composed soils. While labile W was generally greater in the SAND compared to the CLAY soil, the reverse trend was observed in the limed soils at the highest W addition (5000 mg kg-1). Combining our results with previous mechanistic reports suggests that clay edge sorption sites significantly contributed to W retention in treatments with low to medium W additions, resulting in lower environmental availability for W in the CLAY soil. At high W concentrations and high pH, the stronger W retention in the SAND was attributed to continuous formation of W surface polymers on the more abundant metal (oxyhydr)oxides, a process that has been previously reported to occur even under alkaline conditions. A first comparison of various soil chemical methods (Bray & AB-DTPA extractions, soil solution centrifugation CL, diffusion-based DGT) to predict W phytoavailability in soil also revealed a strong pH dependency challenging the identification of a suitable method. This study is one of the first demonstrating the pH dependence of W in natural soils and delivers evidence for increased risk of W mobilization in W polluted, alkaline soil environments.

Arsenic redox transformations and cycling in the rhizosphere of Pteris vittata and Pteris quadriaurita.

Pteris vittata (PV) and Pteris quadriaurita (PQ) are reported to hyperaccumulate arsenic (As) when grown in Asrich soil. Yet, little is known about the impact of their unique As accumulation mechanisms on As transformations and cycling at the soil-root interface. Using a combined approach of two-dimensional (2D), sub-mm scale solute imaging of arsenite (AsIII), arsenate (AsV), phosphorus (P), manganese (Mn), iron (Fe) and oxygen (O2), we found localized patterns of AsIII/AsV redox transformations in the PV rhizosphere (AsIII/AsV ratio of 0.57) compared to bulk soil (AsIII/AsV ratio of ≤0.04). Our data indicate that the high As root uptake, translocation and accumulation from the As-rich experimental soil (2080 mg kg-1) to PV fronds (6986 mg kg-1) induced As detoxification via AsV reduction and AsIII root efflux, leading to AsIII accumulation and re-oxidation to AsV in the rhizosphere porewater. This As cycling mechanism is linked to the reduction of O2 and MnIII/IV (oxyhydr)oxides resulting in decreased O2 levels and increased Mn solubilization along roots. Compared to PV, we found 4-fold lower As translocation to PQ fronds (1611 mg kg-1), 2-fold lower AsV depletion in the PQ rhizosphere, and no AsIII efflux from PQ roots, suggesting that PQ efficiently controls As uptake to avoid toxic As levels in roots. Analysis of root exudates obtained from soil-grown PV showed that As acquisition by PV roots was not associated with phytic acid release. Our study demonstrates that two closely-related As-accumulating ferns have distinct mechanisms for As uptake modulating As cycling in As-rich environments.

Metal solubility in the rhizosphere of a co-cropping system. The role of total carbon exudation, soluble proteins and plant interaction.

In the present study we assessed how modified rhizosphere pH and root exudation (total carbon (C) and soluble proteins released) affected lead (Pb) solubility as well as plant growth and Pb accumulation. A pot experiment with Pb polluted agricultural soils was performed, which involved growing two species, Capsicum annum (pepper) and Tagetes minuta, with the latter being a native herb indicated as potential phytoextractor of Pb, in monocrop and co-cropping conditions. Changes in plant growth, metal uptake as well as rhizosphere soil parameters (pH, EC) and total C and protein exudation were determined. In addition, the metal extraction efficiency of exudates released under mono- and co-cropped conditions were investigated. Results showed that in contrast to the control soil (with low Pb concentration), total C exudation was higher in co-cropping systems in Pb contaminated soils which lead to increases in Pb uptake in both species. Exudates originating from T. minuta were more efficient in solubilizing Pb than exudates from pepper when grown under mono-cropping conditions. Exudates derived from co-cropping both species were either equally or less efficient in mobilizing Pb than exudates from T. minuta. The capacity of exudates to mobilize metals was dependent not only on the species specific quality of root exudates released, but also on its quantity, with the metal extraction efficiency increasing with C concentration in exudates. However, the role of exuded proteins in Pb solubilization was found to be negligible. Biochemical interactions in the rhizosphere under co-cropping conditions favored metal solubilization, and consequently Pb accumulation. The co-cropping conditions could allow accumulation of Pb to levels in pepper that pose risks when the plants are used as a food source.

Molecular Mechanisms of Tungsten Toxicity Differ for Glycine max Depending on Nitrogen Regime.

Tungsten (W) finds increasing application in military, aviation and household appliance industry, opening new paths into the environment. Since W shares certain chemical properties with the essential plant micronutrient molybdenum (Mo), it is proposed to inhibit enzymatic activity of molybdoenzymes [e.g., nitrate reductase (NR)] by replacing the Mo-ion bound to the co-factor. Recent studies suggest that W, much like other heavy metals, also exerts toxicity on its own. To create a comprehensive picture of tungsten stress, this study investigated the effects of W on growth and metabolism of soybean (Glycine max), depending on plant nitrogen regime [nitrate fed (N fed) vs. symbiotic N2 fixation (N fix)] by combining plant physiological data (biomass production, starch and nutrient content, N2 fixation, nitrate reductase activity) with root and nodule proteome data. Irrespective of N regime, NR activity and total N decreased with increasing W concentrations. Nodulation and therefore also N2 fixation strongly declined at high W concentrations, particularly in N fix plants. However, N2 fixation rate (g N fixed g-1 nodule dwt) remained unaffected by increasing W concentrations. Proteomic analysis revealed a strong decline in leghemoglobin and nitrogenase precursor levels (NifD), as well as an increase in abundance of proteins involved in secondary metabolism in N fix nodules. Taken together this indicates that, in contrast to the reported direct inhibition of NR, N2 fixation appears to be indirectly inhibited by a decrease in nitrogenase synthesis due to W induced changes in nodule oxygen levels of N fix plants. Besides N metabolism, plants exhibited a strong reduction of shoot (both N regimes) and root (N fed only) biomass, an imbalance in nutrient levels and a failure of carbon metabolic pathways accompanied by an accumulation of starch at high tungsten concentrations, independent of N-regime. Proteomic data (available via ProteomeXchange with identifier PXD010877) demonstrated that the response to high W concentrations was independent of nodule functionality and dominated by several peroxidases and other general stress related proteins. Based on an evaluation of several W responsive proteotypic peptides, we identified a set of protein markers of W stress and possible targets for improved stress tolerance.

In situ observation of localized, sub-mm scale changes of phosphorus biogeochemistry in the rhizosphere.

AIMS: We imaged the sub-mm distribution of labile P and pH in the rhizosphere of three plant species to localize zones and hot spots of P depletion and accumulation along individual root axes and to relate our findings to nutrient acquisition / root exudation strategies in P-limited conditions at different soil pH, and to mobilization pattern of other elements (Al, Fe, Ca, Mg, Mn) in the rhizosphere. METHODS: Sub-mm distributions of labile elemental patterns were sampled using diffusive gradients in thin films and analysed using laser ablation inductively coupled plasma mass spectrometry. pH images were taken using planar optodes. RESULTS: We found distinct patterns of highly localized labile P depletion and accumulation reflecting the complex interaction of plant P acquisition strategies with soil pH, fertilizer treatment, root age, and elements (Al, Fe, Ca) that are involved in P biogeochemistry in soil. We show that the plants respond to P deficiency either by acidification or alkalization, depending on initial bulk soil pH and other factors of P solubility. CONCLUSIONS: P solubilization activities of roots are highly localized, typically around root apices, but may also extend towards the extension / root hair zone.

pH-Dependent Bioavailability, Speciation, and Phytotoxicity of Tungsten (W) in Soil Affect Growth and Molybdoenzyme Activity of Nodulated Soybeans.

Increasing use of tungsten (W)-based products opened new pathways for W into environmental systems. Due to its chemical alikeness with molybdenum (Mo), W is expected to behave similarly to its "twin element", Mo; however, our knowledge of the behavior of W in the plant-soil environment remains inadequate. The aim of this study was to investigate plant growth as well as W and nutrient uptake depending on soil chemical properties such as soil pH and texture. Soybean ( Glycine max cv. Primus) was grown on two acidic soils differing in soil texture that were either kept at their natural soil pH (pH of 4.5-5) or limed (pH of ≥7) and amended with increasing concentrations of metallic W (control and 500 and 5000 mg kg-1). In addition, the activity of molybdoenzymes involved in N assimilation (nitrate reductase) and symbiotic N2 fixation (nitrogenase) was also investigated. Our results showed that the risk of W entering the food web was significantly greater in high-pH soils due to increased solubility of mainly monomeric W. The effect of soil texture on W solubility and phytoavailability was less pronounced compared to soil pH. Particularly at intermediate W additions (W 500 mg kg-1), symbiotic nitrogen fixation was able to compensate for reduced leaf nitrate reductase activity. When W soil solution concentrations became too toxic (W 5000 mg kg-1), nodulation was more strongly inhibited than nitrogenase activity in the few nodules formed, suggesting a more-efficient detoxification and compartmentalization mechanism in nodules than in soybean leaves. The increasing presence of polymeric W species observed in low-pH soils spiked with high W concentrations resulted in decreased W uptake. Simultaneously, polymeric W species had an overall negative effect on nutrient assimilation and plant growth, suggesting a greater phytotoxicity of W polymers. Our study demonstrates the importance of accounting for soil pH in risk assessment studies of W in the plant-soil environment, something that has been completely neglected in the past.

New Methods To Unravel Rhizosphere Processes.

Root-triggered processes (growth, uptake and release of solutes) vary in space and time, and interact with heterogeneous soil microenvironments that provide habitats for (micro)biota on various scales. Despite tremendous progress in method development in the past decades, finding a suitable experimental set-up to investigate processes occurring at the dynamic conjunction of biosphere, hydrosphere, and pedosphere in the close vicinity of active plant roots still represents a major challenge. We discuss recent methodological developments in rhizosphere research with a focus on imaging techniques. We further review established concepts that have been updated with novel techniques, highlighting the need for combinatorial approaches to disentangle rhizosphere processes on relevant scales.

Environmental impact assessment of wood ash utilization in forest road construction and maintenance--A field study.

The ever increasing use of wood material as fuel for green energy production requires innovative, environmentally safe strategies for recycling of the remaining wood ash. Utilizing wood ash in forest road construction and maintenance to improve mechanical stability has been suggested as a feasible recycling option. To investigate the environmental impact of wood ash application in forest road maintenance, a two-year field experiment was conducted at two Austrian forest sites (Kobernausserwald (KO) (soil pH 5.5) and Weyregg (WE) (pH 7.7)) differing in their soil chemical properties. Two different ashes, one produced by grate incineration (GA) and the other by fluidized bed incineration in a mixture with 15 vol% burnt lime (FBA), were incorporated in repeated road sections at a 15:85% (V/V) ash-to-soil rate. Leaching waters from the road body were collected and analyzed for 32 environmentally relevant parameters over two years. Upon termination of the experiment, sub-road soil samples were collected and analyzed for ash-related changes in soil chemistry. Even though a larger number of parameters was affected by the ash application at the alkaline site (WE), we observed the most pronounced initial increases of pH as well as Al, As, Fe, Mn, Ni, Co, Cu, Mo, and NO2(−) concentrations in leachates beneath GA-treated road bodies at Kobernausserwald due to the lower soil buffer capacity at this site. Despite the observed effects our results indicate that, when specific requirements are met (i.e. appropriate ash quality, sufficient soil buffer capacity below the road body, and single time-point ash incorporation within several decades), wood ash application in forest road construction is generally environmentally acceptable.

O2 dynamics in the rhizosphere of young rice plants (Oryza sativa L.) as studied by planar optodes.

BACKGROUND AND AIMS: Radial O2 loss (ROL) strongly affect the O2 availability in the rhizosphere of rice. The ROL create an oxic zone around the roots, protecting the plant from toxic reduced chemical species and regulates the redox chemistry in the soil. This study investigates the spatio-temporal variability in O2 dynamics in the rice rhizosphere. METHOD: Applying high-resolution planar optode imaging, we investigated the O2 dynamics of plants grown in water saturated soil, as a function of ambient O2 level, irradiance and plant development, for submerged and emerged plants. RESULTS: O2 leakage was heterogeneously distributed with zones of intense leakage around roots tips and young developing roots. While the majority of roots exhibited high ROL others remained surrounded by anoxic soil. ROL was affected by ambient O2 levels around the plant, as well as irradiance, indicating a direct influence of photosynthetic activity on ROL. At onset of darkness, oxia in the rhizosphere was drastically reduced, but subsequently oxia gradually increased, presumably as root and/or soil respiration declined. CONCLUSION: The study demonstrates a high spatio-temporal heterogeneity in rhizosphere O2 dynamics and difference in ROL between different parts of the rhizosphere. The work documents that spatio-temporal measurements are important to fully understand and account for the highly variable O2 dynamics and associated biogeochemical processes and pathways in the rice rhizosphere.