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Subsoils are increasingly studied as they, first, store a great deal of terrestrial carbon (C) and possibly even more, and second, offer resources like water and nutrients to plants, potentially mitigating negative consequences of global change. As subsoil access is often hampered by compacted soil layers, the key to accessing subsoil resources and storing more C below ground might be in biopores. Appropriately nicknamed 'highways of root growth', biopores are macropores left behind by dying roots and earthworm activities, often enriched with organic matter (OM) and nutrients. They are thought to be the most abundant microbial hotspots in the subsoil, thus possibly accounting for a large part of C turnover, as well as offering pore wall nutrients to subsequent crops. Understanding the multifunctionality and complexities of biopores remains challenging. This contribution aims to showcase analytical ways to deepen our understanding of origin and functioning of biopores and hotspots. Regarding their biogeochemistry, biopore OM quality and its turnover can be better unravelled through compound‐specific analysis to deduct biopore‐specific OM turnover. Biopores can be reliably differentiated by their OM quality. A more profound understanding of subsoil C turnover in very contrasting hotspots is crucially important for managing subsoil functions. Biopores are often assumed to be beneficial in crop sequences. Roots making use of specific biopores can be, for the first time, quantified after radiotracer application, two‐step phosphor imaging, and image processing. Combining radioactive with stable isotopes as well as plant and microbial biomarkers allows to investigate the relevance of individual pore wall nutrients in plant growth in consideration of physical biopore properties. Biopore‐friendly management practices (e.g., reduced tillage, perennial cover cropping) could be part of smart subsoil management. Faster access to subsoil water and concentrated biopore nutrients may safeguard agricultural production—especially in times of rising fertiliser costs (both monetary and environmental) and more frequent droughts. [ABSTRACT FROM AUTHOR]

The phospholipid fatty acid (PLFA) composition of soils was analysed at three poplar-based silvo-arable systems and at one willow-based silvo-grassland alley agroforestry system in Central Germany. The objective was to analyse tree row effects on the PLFA composition of main fungal and main bacterial groups. The fungal groups were BAM (Basidiomycota + Ascomycota + Mucoromycota) and AMF (Arbuscular Mycorrhizal Fungi). The bacterial groups were Gram-negative, Firmicutes, and Actinobacteria. The total PLFA content varied between 53 and 170 nmol g−1 soil. Total PLFA and microbial biomass carbon (MBC) showed a strong linear relationship, which resulted in a mean MBC/total PLFA ratio of 4.2 μg nmol−1. AMF contributed on average 4 mol% and the fungal BAM group 10 % to total PLFA. Gram-negative bacteria contributed on average 37 mol%, Firmicutes 23 mol%, and Actinobacteria 6 mol% to total PLFA. The presence of poplar or willow trees increased the mean total PLFA content in comparison with the alleyways by 30 %. Especially the mean contribution of fungal PLFA to total PLFA showed a significant +7.0 mol% increase in the tree row compared with the alleyways, exclusively caused by the BAM group (+7.6 mol%), whereas the contribution of the AMF PLFA linearly decreased from the middle of the alleyway to the tree row at all sites. Within the alleyways, the Gram-negative/Firmicutes PLFA ratio showed a significant decline from the 1 m up to the 24 m distance samples at sites Dornburg and Forst. Despite a decrease of AMF in tree rows, agroforestry tree rows led to a rapid increase in fungi, most likely due to the promotion of ecto-mycorrhizal fungi. • Tree rows shifted the PLFA composition to more fungal biomarkers. • Bacterial PLFA diversity was higher in the arable alleyways than in the tree rows. • PLFA composition contrasts bacterial gene abundance, especially for Actinobacteria. • Agroforestry generally supports the C sequestration potential of mycorrhizal fungi. [ABSTRACT FROM AUTHOR]

Rationale: Many bacteria synthesize carbon (C) and energy storage compounds, including water‐insoluble polyester lipids composed mainly or entirely of poly(3‐hydroxybutyrate) (PHB). Despite the potential significance of C and energy storage for microbial life and C cycling, few measurements of PHB in soil have been reported. Methods: A new protocol was implemented, based on an earlier sediment extraction and derivatization procedure, with quantification by gas chromatography/mass spectrometry (GC/MS) and 13C‐isotopic analysis by GC/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Results: The PHB content was 4.3 μg C g−1 in an agricultural soil and 1.2 μg C g−1 in a forest topsoil. This was an order of magnitude more PHB than obtained by the existing extraction method, suggesting that native PHB in soil has been previously underestimated. Addition of glucose increased the PHB content by 135% and 1,215% over 5 days, with the largest increase in the relatively nutrient‐poor forest soil. In the agricultural soil, 68% of the increase was derived from added 13C‐labeled glucose, confirming synthesis of PHB from glucose for the first time in soil. Conclusions: The presence and responsiveness of PHB in both these contrasting soils show that PHB could provide a useful indicator of bacterial nutritional status and unbalanced growth. Microbial storage could be important to C and nutrient cycling and be a widespread strategy in the life of soil bacteria. The presented method offers new insight into the significance of this compound in soil. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is a major limiting nutrient for plant growth implying an often-intensive competition between microorganisms and plants in the rhizosphere. Increasing the P availability in subsoils may help to mitigate potential future P fertilizer shortages and to overcome P limitations due to droughts, which mainly affect topsoils. Root exudates provide easily available carbon and energy sources for microorganisms to mobilize soil nutrients. Nonetheless, details regarding the distinct processes underlying P mobilization from various P sources (free vs. sorbed PO 4 3−; low molecular vs. complex organic P, e.g. ATP vs. plant litter P) as affected by root exudates are poorly understood, especially in subsoils. This study aimed to identify the controlling factors and microbial processes regulating the availability of organic and inorganic P in top- and subsoils by 33P isotopic labeling. The focus was on the potential key role of root exudates in P mobilization. We found that microbial communities in top- and subsoils used high- and low-available mineral P to a similar extent, but that the subsoil communities were much more efficient in mobilizing and incorporating complex litter-derived organic P. This capability of subsoil communities was even enhanced when root exudates were present. Microbial activity and nutrient-mobilizing mechanisms (e.g., P-related enzymes) clearly increased by root exudate addition, an effect that was generally higher in sub-than in topsoils. We conclude that subsoil communities are well capable of mobilizing and using complex organic P sources, especially if root exudates accelerate overall activity and P cycling. Thus, high root exudation is highly relevant for crops, which depend on subsoil nutrients and litter-derived P. Accordingly, detritusphere P, e.g. in subsoil root channels, is likely to be plant-available because of exudate-induced microbial P (re-)cycling processes. [Display omitted] • Root exudates did not affect growth of the microbial community nor lead to a community shift. • Due to rapid desorption microbes use sorbed P similarly to mineral P added to soil solution. • Low available litter P was mobilized and incorporated more efficient by sub than topsoil communities. • Root exudates increase microbial activity and thus boost P mining process. [ABSTRACT FROM AUTHOR]

Meadows and forests are the main vegetation types in temperate terrestrial ecosystems, and largely contribute to soil carbon (C) stock. Bioavailable C inputs can accelerate microbial decomposition of soil organic matter (SOM), which is known as "priming effect". However, it is still unclear how priming effect, as an important mechanism influencing soil C sequestration, is influenced by spatial transition of vegetation from meadow to forest. To investigate the mechanism of priming effect along a spatial transition gradient of vegetation, a soil incubation experiment with 14C labeled glucose was combined with microbial rDNA sequencing and gene composition prediction. The results showed that with the vegetation transition from meadow to forest soil available phosphorus (P) significantly increased, in contrast to dissolved nitrogen (N) and C which remained unaffected. Moreover, the soil microbial community composition shifted towards a higher relative abundance of K-strategists (Acidobacteria) and a lower abundance of r-strategists (Actinobacteriota) along the vegetation transition from meadow to forest. In the meadow, the microbial community consumed more of the added glucose and increased priming effect. This was accompanied by lower available P but higher soil bacterial gene function encoding P cycling. In contrast, increased soil P availability in forest soils caused a decelerated microbial metabolism of phosphorylated organic compounds within microbial biomass due to decreased microbial demand for P acquisition from SOM, and thus resulted in suppression of the priming effect. Our study showed that P availability and microbial community shifts in spatial transition zones between meadows and forests are important drivers for the priming effect on SOM decomposition. • 14C radioactive isotope labeling was applied in combination with microbial rDNA sequencing. • Soil microbial community in meadow consumed more of the added glucose than that in forest. • Vegetation transition from meadow to forest suppressed the priming effect. • P availability and microbial community shifts are important drivers for the priming effect. [ABSTRACT FROM AUTHOR]

Most subsoil carbon (C) turnover occurs in biopore hotspots such as root channels and earthworm burrows. Biopores allocate large C amounts into the subsoil, where a vast capacity for long-term C sequestration is predicted. We hypothesise that organic matter (OM) cycling in biopores depends on their origin. Earthworm and root biopores were induced under field conditions and were sampled from the subsoil (45–75 and 75–105 cm) after two years of biopore formation. The effects of biopore formation on OM decomposition were studied by biomarkers: neutral sugars, cutin and suberin-derived lipids, lignin-derived phenols and free lipids. The degradation stage of OM was biopore type-specific but was only governed by the soil depth in root biopores. Degradation of OM increased from earthworm biopores to root biopores and bulk soil. Hemicelluloses (GM/AX ratio) were more strongly degraded than lignin side-chains (relative change from initial values). Two years of microbial processing during biopore formation increased the GM/AX ratio in earthworm biopores from 0.65 to 1.05 and in root biopores from 0.15 to 1.35 (both relative to source biomasses). Root biopores and bulk soil had the highest GM/AX ratios (1.2–1.3), hinting to rapid processing of plant residues and accumulation of microbial residues. The regular, frequent OM inputs by earthworms stimulated microbial growth and processing of mostly bioavailable OM and, thus, relatively enriched more persistent OM (e.g. lignin). Syringyl subunits of lignin underwent low (ratio changed from 0.35 to 0.55 relative to initial input) and vanillyl subunits underwent almost no processing in earthworm biopores indicating the preferential microbial utilisation of the easily available compounds frequently replenished by earthworm activity. After two years of decomposition of the root detritus, mainly structural plant material was enriched in root biopores. Short periods (6 months) of earthworm activity effectively recharged the highly processed OM in root biopores with fresh OM. In total, deep-rooting catch crops and short-term earthworm activities promote C accumulation in the subsoil followed by biopore-specific microbial processing predominantly governed by the C input frequency. As root biopores are up to 40 times more common than earthworm biopores, they dominate the OM input into subsoils. Such C inputs create several years lasting hotspots for preferential root growth and nutrient mobilisation in the subsoil. We conclude that root- and earthworm-derived biopores are vertical pathways for plant C from the soil surface into the subsoil and for intensive processing of litter C and sequestration of microbial necromass. [ABSTRACT FROM AUTHOR]

In South-Central Chile, slopes degraded by former erosion have been afforested with exotic tree species since the 1950s for erosion protection. From 1975 on, primary and secondary forests have increasingly been replaced by tree plantations. This practice is often justified by claiming plantations would similarly protect the soil from erosion, even on areas which used to be natural forest. We assessed if plantations offer a comparable level of erosion protection as the natural forests. A six-year-old Eucalyptus globulus (Labill.) plantation was compared to an adjacent secondary forest using soil profiles and erosion proxies, i.e. topsoil parameters linked to erosion and 137 Cs inventories. These pointed to higher erosion in the plantation: the mean bulk density of the plantation was 22% higher compared to the forest site, the gravel content in the plantation was 61% higher, the organic matter content was 20% lower, the mean thickness of the litter layer was 2.2 cm lower and the total sand content was 20% higher. The soil loss of the plantation was estimated to be between 4.8 cm (mean profile truncation) and 6 cm ( 137 Cs approach). These results clearly hint to the conclusion, that the tree plantations in South-Central Chile might promote soil erosion instead of preventing it. Thus, current land use management practices seem to impose erosion risks on Chilean soils raising concern about the sustainable development within the study site. [ABSTRACT FROM AUTHOR]

Root mucilage modulates soil-plant-water dynamics, but its interactions with microbial community functioning remain poorly understood. The aims of this study were to estimate (I) the impacts of mucilage and soil water content on the microbial community composition and (II) the mucilage consumption by individual microbial groups. C4 root mucilage from maize (at 40 and 200 μg C per gram dry soil, corresponding to 10 and 50% of soil microbial biomass, respectively) was added in single pulses to a C3 soil at two moisture levels: optimum (80% of water-holding capacity (WHC)) and drought (30% of WHC). After 15 days of incubation, the microbial community composition was studied by phospholipid fatty acids (PLFA) analysis and incorporation of mucilage-derived C into individual microbial groups was determined by compound-specific isotope analysis. Microbial community composition remained largely unaffected by mucilage addition but was affected by moisture. Whereas an increase in water content reduced mucilage C recovery in PLFA for the low-dose mucilage amendment from 19 to 9%, it had no effect under the high-dose amendment (11-12%). This suggests that the role of mucilage for microbial functioning is especially pronounced under drought conditions. The fungal PLFA 18:2ω6,9 was present only under drought conditions, and fungi profited in their mucilage C utilisation from the lower competitiveness of many bacterial groups under drought. In this study, Gram-negatives (G−, characterised by PLFA 18:1ω9c, 18:1ω7c, 16:1ω7c and cy17:0) showed the highest mucilage-derived C in PLFA, especially at the high-dose amendment, suggesting them to be the major decomposers of mucilage, especially when the availability of this C source is high. Gram-positives (G+) included different sub-groups with distinct responses to moisture: G+ 1 (a15:0) were only competitive for mucilage C under drought, whereas G+ 3 (i17:0) were only able to utilise mucilage-derived C under optimal moisture conditions. During the 15-day incubation, they built up more than 40% of their membranes from mucilage-derived C, suggesting that in the case of high availability, mucilage can act as an important C source for this microbial group. However, under drought, G− 1 and fungi were incorporating the most mucilage C into their membranes (approx. 20% of PLFA-C). The observation that, for some groups, the high-dose mucilage amendments under drought led to higher C incorporation into PLFA than under optimum moisture suggests that mucilage can compensate drought effects for particular microbial groups. Thus, mucilage may not only act as a C source for microorganisms but may also mitigate drought effects for specific rhizosphere microbial groups. [ABSTRACT FROM AUTHOR]

Background and aims: Biopores as microbial hotspots provide additional nutrients to crops - but only if their roots grow within the biopores. Such reuse has never been quantified as pre-crop-specific biopores are hardly differentiated from the multitude of pre-existing biopores. Quantification requires e.g. radionuclide labelling of pre-crops (Cs, to label their biopores) and main crops (C, to detect new roots). Preliminary testing was performed on simulated biopore reuse: both nuclides given to the same plant were excreted into the same rhizosphere. Methods: Cichorium intybus (cv. Puna) and Medicago sativa (cv. Planet) were each sequentially labelled via the leaves with Cs and CO. β-signals were visualised by imaging of horizontal soil cuts - with and without shielding off the weaker C. Results: Both species allocated 7.1-9.4% of the Cs and 21-63% of the C below ground. The first image gave both activities; while the second gave only Cs. Subtracting the second from the first image gave the C distribution, resulting in successful separation of the signals. Thus, separate spatial representations of the roots were obtained. Main root locations by Cs and C showed a very high spatial overlap coefficient (> 0.95). Conclusions: Biopore reuse quantification likely becomes feasible with this sequential labelling and shielding approach. [ABSTRACT FROM AUTHOR]

Biopores are hotspots of nutrient mobilisation and shortcuts for carbon (C) into subsoils. C processing relies on microbial community composition, which remains unexplored in subsoil biopores. Phospholipid fatty acids (PLFAs; markers for living microbial groups) and amino sugars (microbial necromass markers) were extracted from two subsoil depths (45-75 cm ; 75-105 cm) and three biopore types: (I) drilosphere of Lumbricus terrestris L., (II) 2-year-old root biopores and (III) 1.5-year-old root biopores plus six 6 months of L. terrestris activities. Biopore C contents were at least 2.5 times higher than in bulk soil, causing 26-35 times higher Σ PLFAs g soil. The highest Σ PLFAs were found in both earthworm biopore types; thus, the highest soil organic matter and nutrient turnover were assumed. Σ PLFAs was 33% lower in root pores than in earthworm pores. The treatment affected the microbial community composition more strongly than soil depth, hinting to similar C quality in biopores: Gram-positives including actinobacteria were more abundant in root pores than in earthworm pores, probably due to lower C bioavailability in the former. Both earthworm pore types featured fresh litter input, promoting growth of Gram-negatives and fungi. Earthworms in root pores shifted the composition of the microbial community heavily and turned root pores into earthworm pores within 6 months. Only recent communities were affected and they reflect a strong heterogeneity of microbial activity and functions in subsoil hotspots, whereas biopore-specific necromass accumulation from different microbial groups was absent. [ABSTRACT FROM AUTHOR]

Aims: This study investigated the influence of climate and soil on the exudation rate and polysaccharide composition of aerial nodal root mucilage from drought-resistant and drought-susceptible maize varieties. Methods: Two maize varieties were grown in two different soils (sandy-clay loam Acrisol and loam Luvisol) under simulated climatic conditions of their agroecological zones of origin in Kenya and Germany. The exudation rate of mucilage from the aerial nodal roots was quantified as dry weight per root tip per day and the mucilage was characterized for its polysaccharide composition. Results: On average, the mucilage exudation rate was 35.8% higher under the Kenyan semi-arid tropical than under the German humid temperate climatic conditions. However, cultivation in the loam Luvisol soil from Germany led to 73.7% higher mucilage exudation rate than cultivation in the sandy-clay loam Acrisol soil from Kenya, plausibly due to its higher microbial biomass and nutrient availability. The drought-resistant Kenyan maize variety exuded 58.2% more mucilage than the drought-susceptible German variety. On average, mucilage polysaccharides were composed of 40.6% galactose, 26.2% fucose, 13.1% mannose, 11% arabinose, 3.5% glucose, 3.2% xylose, 1.3% glucuronic acid, and 1% an unknown uronic acid. Overall, significantly higher proportions of the uronic acids were found in the mucilage of the plants grown in the Kenyan sandy-clay loam soil and under the Kenyan semi-arid tropical climatic conditions. Conclusions: Maize is able to enhance its mucilage exudation rate under warm climatic conditions and in soils of high microbial activity to mitigate water stress and support the rhizosphere microbiome, respectively. [ABSTRACT FROM AUTHOR]

• Legumes interacted with abiotic site-characteristics affecting soil microbial activity. • Abiotic environmental factors indirectly controlled the basal respiration of microorganisms. • Soybean cultivation helped to overcome C limitation in savannah soils with low C stocks. Unsustainable farming systems for a growing population in Sub-Saharan Africa stress natural resources and lead to soil degradation. Legume cultivation, however, promotes soil microbial communities and may help reverse soil degradation. The soil properties (pH, texture, C and nutrient contents) of a set of contrasting sites from two agro-ecological zones of Cameroon were determined. This study characterises the microbial activities and functioning in rhizosphere soils of the four legumes Phaseolus vulgaris L., Glycine max L., Arachis hypogea L. and Vigna subterranea L. in a laboratory incubation experiment. Ecological indicators (total respiration, microbial biomass determined as total phospholipid fatty acids, metabolic quotient) of soil health were quantified after microbial activation by 14C-glucose and related to site-specific parameters as well as to the effects of legume cultivation. Microbial activation after glucose addition was frequently site-specific. Cumulative glucose respiration increased over time, and glucose pulses repeatedly boosted glucose respiration and total respiration at all sites. The microbial biomass was lowest after the experiment in the soil from the High Guinean Savannah except for soils under soybean cultivation. Among the four legumes cultivated, only soybeans strongly increased microbial biomass in the High Guinean savannah reaching even the level of microbial biomass in Western Highland soils. The lowest metabolic quotient in soybean rhizosphere soil and lowest glucose respiration compared to the soils of other legumes suggests a high carbon use efficiency, presumably due to C limitation in the High Guinean Savannah. Abiotic soil properties (pH, clay content) strongly influenced microbial habitat properties and thus had a positive effect on the CO 2 efflux, irrespective of glucose addition. Within the set of tested legumes, soybean cultivation can, therefore, be strongly recommended as a long-term sustainable crop to boost soil microbial functioning and probably also nutrient cycling, especially under low total organic carbon stocks. [ABSTRACT FROM AUTHOR]

In arable fields, biopores are primarily formed by taproots, but may also be bored by earthworms. Irrespective of the pore origin, repeated use by anecic earthworms yields a wall coating that is rich in carbon, nutrients and microorganisms. However, this effect is halted by routine tillage, and it remains unclear how quickly earthworms are able to alter biopore properties in subsoil. We conducted an earthworm incubation field experiment in arable soil to test the capacity of Lumbricus terrestri s to i. increase total nutrient contents including plant available P, ii. alter the microbial community and iii. increase enzyme activities in biopore walls over one vegetation period. Firstly, biopores that contained chicory roots were identified on a plot scale (4.2 × 1.5 m). After two years under fallow, roots were decomposed. We then inserted individual earthworms at 45 cm depth into a subset of these pores, afterwards refilling with topsoil. After six months, earthworms were removed and soil was opened at 45–75 cm and 75–105 cm soil depth layers. The inner pore wall (1 mm) of individual root biopores (‘RBP’) or root biopores modified by earthworms (‘EBP’) as well as the bulk soil were sampled in 6 depth intervals of 10 cm each and analyzed for total C, N, S content, plant available P, microbial biomass, phospholipid fatty acids (PLFA) and enzyme activity. Biochemical properties of bulk soil, RBP and EBP clearly differed after one vegetation period as indicated by principal component analysis. PLFA markers of fungi and protozoa were detected only in biopores. Compared with the bulk soil, total C, N, S were enriched in RBP by a factor of 2.0–3.1, plant available P by a factor of 8–10, and microbial biomass by a factor of 12–36. In EBP, all of these parameters were as in RBP or elevated even further (C, N, S: factor 1.0–1.4, plant available P: factor 1.3–1.5, microbial biomass: factor 1.5–2.0, PLFA markers of fungi: factor 2.6–4.4, PLFA markers of protozoa: factor 9.2–14.2). PLFA markers indicative of the ratio of Gram-positive to Gram-negative bacteria (G+: G−) were 5–10 fold lower in RBP than in bulk soil, the microbial metabolic quotient ( q CO 2 ) was 0.4–0.6 times as high. In EBP, these parameters were further reduced (ratio G+: G−: factor 0.7, q CO 2 : factor 0.7–0.8). RBP were particularly characterized by high contents of 10-methyl branched fatty acid indicators of actinobacteria. Activities of enzymes involved in the C-cycle (xylanase, cellobiohydrolase, ß-glucosidase) and N-cycle (chitinase, chitotriosidase, leucine aminopeptidase) were also elevated in RBP as compared to the bulk soil (factor 1.1–3.6) and further increased in EBP (factor 1.2–3.7). All these effects were more pronounced in the 45–75 cm soil layer. We conclude that, in only six months, L. terrestris in arable fields modified ordinarily nutrient-rich biopores into ‘super-hotspots’ of microbial biomass, enzyme activity and nutrient availabilities. Hence, even short-term promotion of earthworm populations by agricultural management practices can increase microbial biomass and enzyme activity in biopores and its coupling to nutrient mobilization in the subsoil. [ABSTRACT FROM AUTHOR]

Purpose: Mucilage plays crucial roles in root-soil interactions. Collection systems for maize (Zea mays L.) use primary and seminal roots of aeroponically-grown seedlings (CSA), or brace roots of soil-grown plants (CSB). While each method represents specific plant developmental stages, and root types growing in specific (micro-)environments, these factors are rarely considered. It is unclear whether mucilage exhibits distinct physico-chemical properties related to collection system-inherent factors. Methods: Mucilage of maize genotype B73 was collected from systems CSA and CSB. Chemical composition was assessed by pH, nutrient contents, neutral sugar composition, and polysaccharide polymer length. Viscosity, surface tension and contact angle represented physical properties. Results: The share of hexoses among total polysaccharides was 11% higher in CSB than in CSA, whereas pentoses were predominant in CSA, together with higher nutrient concentrations and pH values. Mannose was detected only in CSB, which also exhibited higher surface tension, viscosity and contact angle compared to CSA. Conclusions: Physico-chemical differences between the two mucilages are related to root type functions, environmental root growth conditions, and plant developmental state. Higher fractions of pentoses in CSA mucilage seem related to semi-sterile system conditions. Higher viscosity of CSB mucilage might reflect the need for enhanced water holding capacity of brace roots growing in drier conditions. A strong influence of environmental factors on mucilage properties even for a single genotype might play additional roles e.g. in the attraction of microbiomes. These aspects are relevant when assessing the role of mucilage in the rhizosphere, or when developing models of rhizosphere processes. [ABSTRACT FROM AUTHOR]

Aims: Visualization of enzymatic activity links microbial functioning to localization in heterogeneous soil habitats. To assess enzymatic reactions in soil thin layer at the microscopic level, we developed a micro-zymography approach and tested it by visualization of the potential activity of phosphomonoesterase for aggregates collected from the rhizosphere of Zea mays L. Methods: We evaluated micro-zymography by applying fluorogenically-labeled substrate i) on individual soil aggregates freshly sampled from the rhizosphere, ii) on thin layers of aggregates (≈ 500 µm) saturated with substrate to assess the dynamics of phosphomonoesterase activity, and iii) on maize roots under laser scanning microscope upon the identified hotspots by membrane-based zymography. Results: We found super transparent silicon as the most appropriate fixative to prevent sample drying. We optimized microscope settings to eliminate the soil auto-fluorescence. The fluorescent signal shifted from the free liquid phase towards the aggregate boundaries within 30 min after substrate addition and was finally detectable at the surface of a few aggregates. This was probably due to higher microbial abundance and enzymatic activity on the soil aggregates compared to the liquid phase. The enzymatic activity appeared patchy at the aggregate and root surfaces indicating heterogeneous distribution of hotspots. Conclusions: The methodology including calibration, sample preparation, fixation, and monitoring was developed. The novel membrane-free micro-zymography approach is a promising tool to identify functional specificity and niche differentiation on roots and soil aggregates. This approach revealed unexplained complexity of competing processes (biochemical, hydrolytic, and physical) due to differently charged reaction products and enzyme-clay complexes. [ABSTRACT FROM AUTHOR]

Comprehensive climate change mitigation necessitates soil carbon (C) storage in cultivated terrestrial ecosystems. Deep-rooted perennial crops may help to turn agricultural soils into efficient C sinks, especially in deeper soil layers. Here, we compared C allocation and potential stabilization to 150 cm depth from two functionally distinct deep-rooted perennials, i.e., lucerne (Medicago sativa L.) and intermediate wheatgrass (kernza; Thinopyrum intermedium), representing legume and non-legume crops, respectively. Belowground C input and stabilization was decoupled from nitrogen (N) fertilizer rate in kernza (100 and 200 kg mineral N ha−1), with no direct link between increasing mineral N fertilization, rhizodeposited C, and microbial C stabilization. Further, both crops displayed a high ability to bring C to deeper soil layers and remarkably, the N2-fixing lucerne showed greater potential to induce microbial C stabilization than the non-legume kernza. Lucerne stimulated greater microbial biomass and abundance of N cycling genes in rhizosphere soil, likely linked to greater amino acid rhizodeposition, hence underlining the importance of coupled C and N for microbial C stabilization efficiency. Inclusion of legumes in perennial cropping systems is not only key for improved productivity at low fertilizer N inputs, but also appears critical for enhancing soil C stabilization, in particular in N limited deep subsoils. [ABSTRACT FROM AUTHOR]

Nitrification acidifies soil, and the produced H+ are neutralized by inorganic carbon (C) in soil leading to irreversible CO 2 emissions. CO 2 released by nitrogen (N) fertilizer-induced acidification is partitioned between solid (CaCO 3 re-precipitation), liquid (dissolved HCO 3 − and CO 3 2−) and gaseous (CO 2) phases. Therefore, quantifying the effects of N fertilization on CO 2 emissions from soil inorganic C is an enormous challenge. 14C-labeled CaCO 3 was used as a model inorganic C to trace the released CO 2 caused by acidification by five fertilizers: chicken manure, urea, KNO 3 , NH 4 NO 3 , and (NH 4) 2 SO 4 added at three N rates. Cropland soil was homogenously mixed with Ca14CO 3 powder and fertilizers, and the emitted CO 2 was trapped in NaOH solution to determine total CO 2 and 14CO 2 efflux originated from inorganic C. Fertilization, particularly ammonium-based fertilizers ((NH 4) 2 SO 4 , NH 4 NO 3), strongly decreased soil pH by 0.35 units over 40 days. All fertilizers except KNO 3 increased total CO 2 emissions by 21%–490% compared to the unfertilized control soil. The fertilization effects on cumulative 14CO 2 emission induced by CaCO 3 neutralization, corresponded to acidification and decreased in the order (NH 4) 2 SO 4 > NH 4 NO 3 > urea > KNO 3 > chicken manure. Ammonium-based fertilizers induced the strongest CO 2 emissions originated from inorganic C, emitting 1.6–4.5 times more 14CO 2 than non-fertilized soils during the first nine days. The total CO 2 emissions from SIC were proportional to the fertilizer dose applied. Therefore, we conclude that both the choice of N fertilizers and their application rates need to be considered to control CO 2 emissions originated from inorganic C. The soil inorganic C losses should be prevented not only because of their irreversible contribution to atmospheric CO 2 , but also to safeguard ecosystem services of CaCO 3 , such as organic matter preservation, soil structure stabilization, and C sequestration. • 14C-labeled CaCO 3 applied to quantify CO 2 emission from soil inorganic carbon (SIC). • CO 2 originated from SIC (CO 2-SIC) increases with nitrification rate of N fertilizers. • Theoretical estimations based on nitrification of N fertilizers overestimates CO 2-SIC emission from soil. • Ignoring the contribution of SIC to total soil CO 2 emission overestimates SOC-originated CO 2. [ABSTRACT FROM AUTHOR]

Alternate wetting-drying (AWD) in rice cultivation controls soil redox conditions and consequently nutrient solubility. Under low redox potential, ferric iron reduction leads to bound phosphate (Fe(III)–P) dissolution, but lack of oxygen retards organic phosphorus (P org) mineralization. Microorganisms accelerate P org mineralization as the redox potential increases during drying, but it is not known which P source will be preferentially taken up by microorganisms and plants. Using double 32/33P labeling, we traced for the first time the P released from inorganic (32P) and organic (33P) sources into microbial biomass P (MBP), phospholipids, and plants under continuous flooding (CF) and AWD. The CF rapidly induced reducing conditions that increased Fe–P dissolution and thus P availability. The AWD had no preference for 32P and 33P incorporation into plants. However, the ratios of 32P in roots to 32P in MBP or to 32P in phospholipids in rooted soil were 4–9 times higher than the respective ratios of 33P. Moreover, the ratios of 33P in roots to 33P in MBP or to 33P in phospholipids were always <0.6. Thus, plants and microorganisms were more competitive for P from Fe–P and from P org , respectively. The AWD increased 32P and 33P incorporation into phospholipids in rooted soil by 53–56% as compared to CF. Also, the ratio of 32P in phospholipids to 32P in MBP was 1.4–2.2 times higher under AWD than under CF. Thus, AWD stimulated microorganisms to allocate more P for cell membrane synthesis as compared to CF. Plants were similarly effective to take up P from inorganic and organic sources. Increased root biomass stimulated arbuscular mycorrhizal symbiosis (represented by 16:1ω5c) and thus 33P recovery in roots under AWD. In turn, the acceleration of microbial P turnover and the microbe-specific preference for P org highlight the importance of arbuscular mycorrhiza for plant P uptake under fluctuating water conditions. [Display omitted] • Continuous flooding increased reductive dissolution of Fe–P and thus P availability. • Microbes allocated more P for cell membrane synthesis under alternate wetting-drying. • Microorganisms in a maintenance state allocated more P for cell compound synthesis. • Rice plants were similarly effective to take up P from inorganic and organic sources. [ABSTRACT FROM AUTHOR]

Plastics accumulating in the environment, especially microplastics (defined as particles <5 mm), can lead to a range of problems and potential loss of ecosystem services. Polyhydroxyalkanoates (PHAs) are biodegradable plastics used in mulch films, and in packaging material to minimize plastic waste and to reduce soil pollution. Little is known, however, about the effect of microbioplastics on soil-plant interactions, especially soil microbial community structure and functioning in agroecosystems. For the first time, we combined zymography (to localize enzyme activity hotspots) with substrate-induced growth respiration to investigate the effect of PHAs addition on soil microbial community structure, growth, and exoenzyme kinetics in the microplastisphere (i.e. interface between soil and microplastic particles) compared to the rhizosphere and bulk soil. We used a common PHAs biopolymer, poly (3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV) and showed that PHBV was readily used by the microbial community as a source of carbon (C) resulting in an increased specific microbial growth rate and a more active microbial biomass in the microplastisphere in comparison to the bulk soil. Higher β-glucosidase and leucine aminopeptidase activities (0.6–5.0 times higher V max) and lower enzyme affinities (1.5–2.0 times higher K m) were also detected in the microplastisphere relative to the rhizosphere. Furthermore, the PHBV addition changed the soil bacterial community at different taxonomical levels and increased the alpha diversity, as well as the relative abundance of Acidobacteria and Verrucomicrobia phyla, compared to the untreated soils. Overall, PHBV addition created soil hotspots where C and nutrient turnover is greatly enhanced, mainly driven by the accelerated microbial biomass and activity. In conclusion, microbioplastics have the potential to alter soil ecological functioning and biogeochemical cycling (e.g., SOM decomposition). [Display omitted] • Microplastisphere (soil-MPs interface) is localized and visualized by zymography. • MPs stimulates microbial turnover and nutrient efficiency in microplastisphere. • MPs increases soil enzyme activity and shifts bacterial community to K -strategy. • MPs have the potential to alter soil functioning and biogeochemical cycling. [ABSTRACT FROM AUTHOR]

Root morphology reflects plant adaptations to phosphorus (P) deficiency. We hypothesized that changes in rice root morphology reflect P deficiency decrease after ferric iron (Fe(III))-bound phosphate (Fe–P) dissolution in low-redox paddy soils. We developed a novel in-situ 32P phosphor-imaging approach under flooding to estimate P uptake by rice roots released from Fe–P dissolution. 32P-labeled ferrihydrite (31 mg P kg−1) was supplied either (1) in polyamide mesh bags (30 μm mesh size) to prevent roots but not microorganisms from direct Fe–P mobilization, or (2) directly mixed with soil to enable roots and microorganisms unrestricted access to the Fe–P. The establishment of low redox conditions (E h values between −176 and −224 mV) drove the reductive dissolution of Fe–P. Rice root-derived organic acids alone were unable to control Fe–P dissolution, and Fe(III) reduction is predominately a microbially-mediated process. Direct root access to Fe–P raised both the number and mean diameter of crown roots and root tips, and increased P uptake by 149–231%. Crown root elongation rate, 32P activities along roots and root tips were 5–133% higher when roots directly accessed Fe–P compared to Fe–P excluded from roots in mesh bags. Iron accumulation on roots depended on the rice growth stage, but not on their access to Fe–P. Roots' access to Fe–P increased rice crown roots elongation and branching and increased P accessibility under P deficiency. [Display omitted] • Fe–P dissolution increased rice root growth and induced root morphological changes. • Increased root tip numbers raised P uptake by roots from the soil. • Organic acids exuded by rice roots were unable to control Fe–P dissolution. • Iron accumulation on root surface was independent of root's access to Fe–P. [ABSTRACT FROM AUTHOR]

Biopores are pores or voids in soil produced by roots, by earthworms, or by the occupation of earthworms in root pores, which are considered important microbial hotspots, especially in subsoil. We hypothesized that earthworms ( Lumbricus terrestris L.) exert stronger effects on microbial activities than decaying plant roots (of Cichorium intybus L.) in the subsoil because of the addition of pre-digested organic material. We tested this hypothesis by analyzing microbial biomass (C), total organic C (C), and activities of eight enzymes (cellobiohydrolase, β-glucosidase, xylanase, acid phosphomonoesterase, leucine aminopeptidase, tyrosine aminopeptidase, chitotriosidase, and n-acetylglucosaminidase) down to 105-cm depth. The C increase was associated with a two- to threefold increase of C content in biopores as compared to bulk soil. The highest percentage of C-to-C (3.7 to 7.3 %) in the drilosphere demonstrated the enhancement of microbial efficiency for organic matter decomposition by earthworms. The availability of organic matter in biopores increased the activities of C- and N-targeting enzymes by 1.2-11.3 times, but reduced acid phosphomonoesterase activity by 10-40 % in biopores versus bulk soil. Introducing earthworms in root biopores caused 1.5-1.8 times higher microbial biomass and 1.2-1.9 times increased enzyme activities compared to the sole effect of earthworms. Soil depth showed a strong effect on the drilosphere, but only slight effects on the biochemical properties of root biopores and bulk soil. In conclusion, biopores are important microbial hotspots of C, N, and P transformations in subsoil. Earthworms exerted stronger effects on biochemical properties of biopores than decaying roots. [ABSTRACT FROM AUTHOR]

Biogeochemical cycles of phosphorus (P) and iron (Fe) are tightly interlinked, especially in highly weathered acidic subtropical and tropical soils rich in iron (oxyhydr)oxides (Fe(III)). The quantitative contribution of the reductive dissolution of Fe(III)-bound inorganic P (P i) (Fe–P) in low-redox paddy soils may cover the demands of rice plants (Oryza sativa L.) and microorganisms. We hypothesized that microbially-driven Fe(III) reduction and dissolution can cover the P demand of microorganisms but not of the young rice plants when the plants' P demand is high but their root systems are not sufficiently developed. We grew pre-germinated rice plants for 33 days in flooded rhizoboxes filled with a paddy soil of low P availability. 32P-labeled ferrihydrite (30.8 mg kg−1) was supplied either (1) in polyamide mesh bags (30 μm mesh size) to prevent roots from directly mobilizing Fe–P (pellets-in-mesh bag treatment), or (2) in the same pellet form but without a mesh bag to enable roots accessing the Fe–P (pellets-no-mesh bag treatment). Without mesh bags, P i was more available leading to increases in microbial biomass carbon (MBC) by 18–55% and nitrogen (MBN) by 4–108% in rooted soil as compared to P i pellets not directly available to roots. The maximum enzyme activities (V max) of phosphomonoesterase and β-glucosidase followed this pattern. During rice root growth, day 10 to day 33, MBC and microbial biomass phosphorus (MBP) contents in both rooted and bottom bulk (15–18 cm) soil gradually decreased by 28–56% and 47–49%, respectively. In contrast to our hypothesis, the contribution of Fe–P to MBP remarkably decreased from 4.5% to almost zero from 10 to 33 days after rice transplantation, while Fe–P compensated up to 16% of the plant P uptake at 33 days after rice transplantation, thus outcompeting microorganisms. Although both plants and microorganisms obtained P i released by Fe(III) reductive dissolution, this mechanism was not sufficient for the demand of either organism groups. [Display omitted] • 32P application can assess plant-microbial competitive dynamics for P absorbed by Fe. • Contribution of Fe–P to MBP decreased from 4.5% to almost zero with rice growth. • Fe–P compensated up to 16% of rice P uptake during 33 days after rice transplantation. • Rice plants outcompeted microorganisms for P at the tillering stage. [ABSTRACT FROM AUTHOR]

Plants stimulate microbial enzyme production in the rhizosphere, regulating soil organic matter decomposition and nutrient cycling. The availability of labile organic compounds (i.e. exudates) and water is the main prerequisite for such microbial activity and enzyme production, thus shaping the rhizosphere. Root morphology (i.e., root hairs) and exudate composition define the spatial distribution of properties and functions in the rhizosphere. However, the role of root architecture and exudate composition in this spatial self-organization of the rhizosphere remains unknown. We coupled three in situ imaging approaches: 14C imaging to localize exudates, soil zymography to analyze enzyme activity distribution, and neutron radiography for water fluxes to trace the spatial structure of the rhizosphere of three maize genotypes (wild-type, mutant with defective root-hair prolongation rth3 , and mutant with reduced benzoxazinoid content in root exudates bx1). The co-localization of these three soil images revealed the pivotal role of both optimal water content (neutron radiography) and root exudation (14C imaging) for β-glucosidase production by the rhizosphere microbiome and its hydrolytic activity (zymography). Root hairs increased the exudate release and enlarged the spatial extent of increased β - glucosidase activity around the root axis by 35%, leading to a two-fold faster decomposition of 14C exudates compared to the mutant with defective root hairs. In contrast, benzoxazinoids suppressed β - glucosidase activity by 30%, reflecting decreased microbial activity, whereas their absence broadened the rhizosphere. Overall, root hairs in wild-type maize increased microbial activity (i.e. β - glucosidase production), whereas the benzoxazinoids in root exudates suppressed microorganisms. [Display omitted] • Zymography, 14C imaging, and neutron radiography were coupled to localize rhizosphere processes. • Exudates are released at root tip in wild-type maize, but along the whole root in rth3 and bx1 mutants. • Root hairs enlarged the spatial extent of increased β - glucosidase activity around the root by 35%. • Benzoxazinoids (secondary metabolites) suppressed β - glucosidase activity in the rhizosphere by 30%. • Hotspot co-localization revealed that exudates and water increased β - glucosidase activity. [ABSTRACT FROM AUTHOR]

Despite the importance of subsoil carbon (C) deposition by deep-rooted crops in mitigating climate change and maintaining soil health, the quantification of root C input and its microbial utilization and stabilization below 1 m depth remains unexplored. We studied C input by three perennial deep-rooted plants (lucerne, kernza, and rosinweed) grown in a unique 4-m deep RootTower facility. 13C multiple pulse labeling was applied to trace C flows in roots, rhizodeposition, and soil as well as 13C incorporation into microbial groups by phospholipid fatty acids and the long-term stabilization of microbial residues by amino sugars. The ratio of rhizodeposited 13C in the PLFA and amino sugar pools was used to compare the relative microbial stability of rhizodeposited C across depths and plant species. Belowground C allocation between roots, rhizodeposits, and living and dead microorganisms indicated depth dependent plant investment. Rhizodeposition as a fraction of the total belowground C input declined from the topsoil (0–25 cm) to the deepest layer (360 cm), i.e., from 35%, 45%, and 36%–8.0%, 2.5%, and 2.7% for lucerne, kernza, and rosinweed, respectively, where lucerne had greater C input than the other species between 340 and 360 cm. The relative microbial stabilization of rhizodeposits in the subsoil across all species showed a dominance of recently assimilated C in microbial necromass, thus indicating a higher microbial stabilization of rhizodeposited C with depth. In conclusion, we traced photosynthates down to 3.6 m soil depth and showed that even relatively small C amounts allocated to deep soil layers will become microbially stabilized. Thus, deep-rooted crops, in particular lucerne are important for stabilization and storage of C over long time scales in deep soil. Image 1 • Rhizodeposition strongly declined with depth across all plant species. • Microbial stabilization of rhizodeposits increases with depth. • Deep-rooted crops are important for storage and stabilization of rhizodeposition. [ABSTRACT FROM AUTHOR]

Despite detailed investigations on the distinct biochemical properties of the rhizosphere and their effects on the availability of nutrients for plants, their biophysical aspects, particularly the effect of mucilage on the transport of water and nutrients are poorly understood. The aim of this study was to investigate the effect of mucilage on the diffusion coefficient of nutrients and consequently their transport in the soil and into the plant roots. Repeated phosphor imaging was used to monitor the temporospatial distribution of 137Cs (as an analog of K) within a modeled rhizosphere soil with and without mucilage (a sandy soil amended with mucilage extract from chia seed) under different soil water contents. The monitored profiles of activities were used to estimate the diffusion coefficient of soils with and without mucilage by solving a diffusion equation. Assuming an identical the effect of mucilage on diffusion coefficient of Cs and K, a diffusion-convection equation was numerically solved to predict the transport of K within the soil and its uptake by a single plant root during a soil drying cycle. To this end, the hydraulic and diffusive properties of the soil were parameterized based on the measured data and the K uptake and its concentration in soil were taken from literature data. The results of this study suggest that mucilage in the rhizosphere keeps the rhizosphere wetter and maintains the connectivity of the liquid phase during a soil drying cycle, and thereby could prevent a marked drop in the diffusion coefficient. The results of modeling of nutrient uptake by a single root showed that the presence of mucilage in the rhizosphere could favor nutrient uptake by the plant root. In the case of nutrients with low concentration in the soil solution, it prevents a marked concentration drop in the vicinity of the root as the soil dries and diffusion becomes restricted. This will delay the risk of nutrient deficiency to the plant root. In the case of nutrients with a high concentration in the soil solution, the presence of mucilage in the rhizosphere may delay the risk of salinity stress as the soil dries and the concentration of nutrients increases at the vicinity of the root surface. In conclusion, the results of this contribution show that mucilage may favor the transport of nutrient within the soil and their uptake by plant roots under drying soil condition. [ABSTRACT FROM AUTHOR]