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Straw return strategies are widely used green management practices that can alter soil organic matter transformation and dynamics through changes in microbial community structure and functions. How the exogenous input of organic materials of contrasting qualities affects the composition of dominant taxa, growth, and microbial functional properties related to nutrient acquisition in space remains unclear. In this study, we investigated the hotsopts and kinetics of C- and N-acquiring hydrolases, microbial growth, and bacterial community structure in maize rhizosphere hotspots after the addition of straw and straw-derived biochar using soil zymography, substrate-induced respiration and high-throughput sequencing. Compared with no amendment and maize straw-derived biochar, straw addition increased the growing biomass and microbial specific growth rate by 1.2–1.6 and 1.7–2.0-fold, respectively, indicating the relative dominance of fast-growing r-strategists. This corresponds to an increased relative abundance of the keystone taxa Firmicutes and their gene copies encoding β-1,4-glucosidase (BG) and β- N -acetylglucosaminidase (NAG). The potential activity and affinity (V max and K m) of BG increased 2.2 and 1.8 times, respectively, and those of NAG increased 4.0 and 2.0 times, respectively. In contrast, the relative abundance of Actinobacteria belonging to K- strategists increased in the biochar-amended soil. This resulted in slower growth and retarded enzymatic activity than the straw return treatment. Biochar enhanced the root biomass by 31% and increased the rhizosphere hotspot extents of BG and NAG by 26% and 47%, respectively. The highest robustness and modularity of the co-occurrence network indicated a more stable network with biochar input. In summary, the addition of straw accelerated rhizosphere nutrient cycling by triggering microbial growth, especially fast-growth r -strategists (Firmicutes), and synthesizing a large number of enzymes. In contrast, the addition of biochar increased rhizosphere nutrient mobilization by expanding the extent of rhizosphere hotspots to mobilize nutrients from a larger soil volume. This suggests that there are different strategies for nutrient mobilization in the rhizosphere with contrasting exogenous C addition. [Display omitted] • Straw addition increased V max and K a of the hydrolase in rhizosphere hotspot. • Straw addition stimulated microbial growth corresponding to increased r -strategists. • Biochar addition expanded the extents of rhizosphere hotspot of BG and NAG. • Biochar addition increased co-occurrence stability and triggered key-taxa with K -strategy. [ABSTRACT FROM AUTHOR]

The soil volume affected by roots – the rhizosphere – is one of the most important microbial hotspots determining the processes, dynamics and cycling of carbon (C), nutrients and water in terrestrial ecosystems. Rhizosphere visualization is necessary to understand, localize and quantify the ongoing processes and functions, but quantitative conclusions are very uncertain because of: 1) the continuum of the parameters between the root surface and root-free soil, i.e., there are no sharp borders, 2) differences in the distributions of various parameters (C, nutrients, pH, enzyme and microbial activities, gases, water etc.) across and along roots, 3) temporal changes of the parameters and processes with root growth as well as with water and C flows. In situ techniques: planar optodes, zymography, sensitive gels, 14C and neutron imaging as well as destructive approaches (thin layer slicing) have been used to analyze the rhizosphere extent and the gradients of various physico-chemical and biological characteristics: pH, CO 2 , O 2 , redox potential, enzyme activities, content of water, nutrients and excess elements, and organic compounds. A literature analysis allows the conclusion that: i) the rhizosphere extent for most of the parameters assessed by non-destructive visualization techniques is 0.5–4 mm but exceeds 4 mm for gases, nitrate, water and redox potential. ii) The rhizosphere extent of nutrients (N, P) is decoupled from the extent of the corresponding enzyme activities. iii) The imbalance between element flows to and uptake by roots may lead to accumulation of excess elements and formation of root carapaces (e.g. CaCO 3 rhizoliths, Fe plaque) ranging up to a few cm. iv) All destructive approaches show a much (3–5 times) larger rhizosphere extent compared to visualization techniques. These conclusions are crucial for a mechanistic understanding of rhizosphere properties and functioning, estimation of the nutrient stocks available to roots, and for rhizosphere modelling considering root growth and architecture. Overall, roots function as ecosystem engineers and build their environment, serving as the main factors shaping rhizosphere extent. Sharp gradients are formed within a few days for nutrients and enzymes, but more time is necessary for the establishment of specific microbial communities. Despite the very strong dynamics of most parameters, their stationarity is reached within a few days because the release of C and enzymes or nutrient uptake are very quickly compensated by utilization by surrounding microorganisms or/and sorption and diffusion processes. We conclude that despite the dynamic nature of each property, the rhizosphere gradients, their extent and shape are quasi-stationary because of the opposite directions of their formation processes. Image 1 • The rhizosphere size and shape were summarized based on gradients of various physical, chemical and biological properties. • The rhizosphere extent for most of the parameters assessed by non-destructive visualization techniques is 0.5–4 mm. • Sharp gradients are formed within few days for nutrients and enzymes; longer periods are necessary for microbial communities. • The rhizosphere gradients are dynamic but quasi-stationary because of the opposite directions of their forming processes. • The stationarity of most parameters is reached within a few days. [ABSTRACT FROM AUTHOR]

Global warming increases belowground carbon (C) input as plant litterfall, root biomass and rhizodeposition, which influences the stocks and dynamics of soil organic matter. To clarify the effects of labile C availability (biochemical factor) and temperature (environmental factor) on enzyme activities, we incubated typical paddy soil for 75 d at four temperatures (5, 15, 25, and 35 °C) under anaerobic conditions. Acetate was used as the source of labile C and methane. The potential activities of three hydrolases (β -glucosidase, chitinase, and xylanase) were analysed on days 3, 15, and 75 after acetate addition. Activity of β -glucosidase and chitinase in soil without acetate addition was 2.1–2.7 times higher than that with acetate. Xylanase activity increased with temperature and incubation period. The enzymes involved in the C cycle were sensitive to temperature, whereas chitinase (responsible for N cycle) activity became temperature sensitive only after acetate addition (Q 10 - V max ≥ 1). Organic C mineralisation (CO 2 release) was more sensitive at low temperature with Q 10 values 1.1–3.4 times higher at 5–15 °C than at 25–35 °C. The Q 10 values for methane (CH 4) emission were 2.8–13.5 times higher at 5–15 °C than at 25–35 °C. Organic matter decomposition in paddy soil was more sensitive to temperature (Q 10 of CO 2 and CH 4 emission ≥ 1) than enzyme activities. Comparison of abiotic (temperature) and biochemical (C availability) effects indicated that warming has limited effects on hydrolase activities in paddy soil. The increase in labile C remarkably stimulated microbial activity and soil organic matter turnover. We conclude that: i) enzyme activities are more sensitive to C addition than to temperature; ii) and SOM decomposition is accelerated by both C input and warming, especially at low temperatures. Image 1 • Labile C affects the response of extracellular enzyme activities to soil warming. • The higher the labile C content, the less enzymes are produced by soil microorganisms. • Labile C addition affects microbial biomass and induces enzymatic N mining from SOM. • Acetate input increases temperature sensitivity of CO 2 and CH 4 emission below 15 °C. [ABSTRACT FROM AUTHOR]

As the primary microbial substrate after shoot cutting, the element stoichiometry of root-detritus (dying or dead roots) influences the enzyme activity in root-detritusphere. However, the effect of the C/P ratio of root-detritus on the dynamics and distribution of enzyme activities is little revealed. We hypothesised that P fertilisation would decrease the C/P ratio of root-detritus, therefore affecting the hotspot areas and hot moments of C-acquiring and P-acquiring enzyme activities, as well as their activity ratio (C/P acquisition ratio). Root-detritus of low (59.0) and high (170.8) C/P ratios was produced in P-poor soil with and without P fertilisation, respectively. In situ soil zymography showed that the distribution of C-acquiring enzymes (β-glucosidase and cellobiohydrolase) was more associated with root-detritus than P-acquiring enzymes (acid and alkaline phosphomonoesterase). P fertilisation increased the hotspot areas of C-acquiring enzyme activities over the experiment, without influencing their temporal dynamics. However, its effect on phosphomonoesterase activities depended on the decomposition and delayed the appearance of the highest hotspot areas. P supply met the microbial demand in P-fertilised soil, with high C/P acquisition ratio and constant stoichiometry of microbial biomass C (MBC)/microbial biomass P (MBP). A low C/P acquisition ratio and high MBC/MBP in non-fertilised soil was observed, indicating P limitation for microorganisms. After the 150-day incubation, Olsen P significantly increased in P-fertilised soil (P < 0.05), whereas it decreased in the root-detritusphere of non-fertilised soil. We conclude that the decomposition of root-detritus with a low C/P ratio has potential to improve soil P availability; however, C-P imbalance may increase during the decomposition of root-detritus with a high C/P ratio. [ABSTRACT FROM AUTHOR]

Nematodes are among the most important pathogens in agriculture, greatly reducing crop biomass and yield. The direct effects of nematodes on above– and belowground plant parts are well known, but the broad range of indirect effects, especially on carbon (C) and phosphorus (P) cycles underground, remains unknown. For the first time, using soil zymography, we analyzed the indirect effects of Meloidogyne incognita cellobiohydrolase and phosphatase. The rhizosphere of lupine ( Lupinus polyphyllus L.), a species sensitive to pathogens with high P demand, was selected to study the activity, distribution and localization of two enzymes responsible for C and P cycling: The distribution patterns of cellobiohydrolase and phosphatase demonstrated that M. incognita induced the formation of knots as well as cluster roots, which corresponded to hotspot locations on zymogram images for both enzymes. Increased C release by nematode–infected roots into the soil led to a decrease in the overall activity of cellobiohydrolase and especially at hotspots (by ∼ 20 times). In contrast, the increased P demand of infected plants raised the phosphatase activity, leading to an increase in the rhizosphere extent around the roots and especially of the hotspot area (by 6 times). Remarkably, this 1 mm increase of rhizosphere extent in 2D equals a 2-fold increment in soil volume (3D) for nutrient mobilization. We conclude that nematode infection not only has direct effects by changing root morphology, but also induces a number of subsequent biochemical changes (e.g. enzyme activities and consequently nutrient mobilization) in the rhizosphere, affecting C and P cycling. [ABSTRACT FROM AUTHOR]

Manure is an important source of nutrients for plants and stimulates a wide range of enzyme-mediated microbial processes. Such stimulation, however, depends on manure distribution and the duration of its decomposition in soil. For the first time, we investigated the spatio-temporal patterns of enzyme activities as affected by manure application strategies: 1) Localized manure: manure application as a layer in the upper soil; 2) Homogenized manure: mixing manure throughout the soil; and 3) Control without manure. Tibetan barley was planted on soil managed with yak manure from the Tibetan Plateau. Soil zymography was used to visualize the two-dimensional distribution and dynamics of the activities of three enzymes responsible for cycling of carbon (β-glucosidase), nitrogen (N-acetylglucosaminidase) and phosphorus (phosphomonoesterase) over 45 days. The manure detritusphere increased enzyme activities relative to the control (which had only the rhizosphere effect of barley) and this stimulation lasted less than 45 days. Enzyme activities in the manure-induced hotspots were higher than on the barley rhizoplane, indicating that the detritusphere stimulated microbial activities more strongly than roots. Homogenized manure led to 3–29% higher enzyme activities than localized manure, but shoot and root biomass was respectively 3.1 and 6.7 times higher with localized manure application. Nutrients released by high enzyme activities within the whole soil volume will be efficiently trapped by microorganisms. In contrast, nutrients released from manure locally are in excess for microbial uptake and remain available for roots. Consequently, microorganisms were successful competitors for nutrients from homogeneous manure application, while plants benefited more from localized manure application. We conclude that localized manure application decreases competition for nutrients between the microbial community of manure and the roots, and thereby increases plant performance. [ABSTRACT FROM AUTHOR]

High latitude and cold ecosystems, which constitute the major environment on Earth, are particularly threatened by global warming. Consequently, huge amounts of SOC stored in these ecosystems may be released to the atmosphere by accelerated enzymatic decomposition. Effects of intensive warming on temperature sensitivity and catalytic properties of soil enzymes were tested in cold-adapted alpine grassland of the Tibetan Plateau. We hypothesized that 1) maximal reaction rate will be insensitive to intensive warming at high temperature range ( V max − Q 10 = 1); 2) substrate affinity ( K m ) remains constant at elevated temperatures due to expression of enzymes with less flexibility. These hypotheses were tested by examining the kinetics of six enzymes involved in carbon (cellobiohydrolase, β-glucosidase, xylanase), nitrogen (tyrosine-aminopeptidase, leucine-aminopeptidase) and phosphorus (acid phosphomonoesterase) cycles after soil incubation at temperatures from 0 to 40 °C. Q 10 and E a decreased at high temperature (25–40 °C). However, enzymes that degrade low quality polymers remained temperature-sensitive even above 25 °C ( V max − Q 10 = 2), which explains the faster decomposition of recalcitrant C compounds under warming. Substrate affinity of all enzymes gradually increased up to 20 °C. At 25 °C, however, K m increased rapidly, leading to an extreme decrease in catalytic efficiency. Above 25 °C, K m of C and N cycles remained nearly constant, while V max gradually increased from 0 to 40 °C. These results reveal two important implications of warming: 1) there are some temperature thresholds (here 20–25 °C) that lead to sudden reductions in substrate affinity, decreasing temperature sensitivity and catalytic efficiency, 2) decoupled temperature sensitivity of V max and K m and the resulting maintenance of stable enzyme systems at high temperatures ensured efficient enzymatic functioning and persistent decomposition of SOM at temperatures much higher than the common adaptation range of the ecosystem. Thus, the temperature thresholds of strong changes in enzyme-based processes should be considered and included in the next generation of models in order to improve the prediction of SOM feedbacks to warming. [ABSTRACT FROM AUTHOR]

Earthworms boost microbial activities and consequently create hotspots in soil. Although the presence of earthworms is thought to change the soil enzyme system, the distribution of enzyme activities inside worm burrows is still unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside worm burrows (biopores) by in situ soil zymography. Kinetic parameters ( V max and K m ) of 6 enzymes – β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) – were determined in pores formed by Lumbricus terrestris L. In earthworm burrows, the spatial distributions of GLU, NAG and APT become observable in zymogram images. Zymography showed a heterogeneous distribution of hotspots in the rhizosphere and worm burrows. The hotspot areas were 2.4–14 times larger in the burrows versus reference soil (soil without earthworms). The significantly higher V max values for GLU, CBH, XYL, NAG and APT in burrows confirmed that earthworms stimulated enzyme activities. For CBH, XYL and NAG, the 2- to 3-fold higher K m values in burrows indicated different enzyme systems with lower substrate affinity compared to reference soil. The positive effects of earthworms on V max were cancelled by the K m increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g −1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the reference soil. We conclude that earthworm burrows are microbial hotspots with much higher and denser distribution of enzyme activities than reference soil. [ABSTRACT FROM AUTHOR]

Microbial parameters have been used to monitor changes in soil quality. Soils from four land use systems common in East Africa and present in the Mt. Kilimanjaro region: (1) montane forest, (2) savannah (3) maize fields and (4) Chagga homegardens were used in laboratory incubations to assess the effects of landuse changes on soil quality. Soil organic matter mineralization and the following microbial parameters: microbial biomass C, mineralization quotient, metabolic quotient and activities of four enzymes: β-glucosidase, cellobiohydrolase, phosphatase and chitinase were determined. Microbial biomass C content, β-glucosidase, cellobiohydrolase and chitinase activities were higher in natural systems compared to agricultural soils. High phosphatase activity observed in all land use types reflected strong phosphorus limitation in andic soils of the Mt. Kilimanjaro region. Chitinase activity in montane forest soils was 3 times higher than in Chagga homegardens. Mineralization quotient and cellobiohydrolase activity best exhibited the effect of land-use changes on soil quality in the Mt. Kilimanjaro region. Cellobiohydrolase activity was up to 3 times higher under natural ecosystems compared to agroecosystems. A high percentage of microbial biomass C content in total organic C and low metabolic quotient were observed in Chagga homegarden soils. Soil enzymes (especially cellobiohydrolase) best distinguished between natural and agricultural ecosystems, and are therefore useful for monitoring changes in soil quality. In conclusion, the measured microbial parameters clearly show that the microbial organisms in traditional Chagga homegardens system have high substrate use efficiency. This demonstrates that traditional agroforestry systems promotes soil fertility and are more suitable for agricultural production in the tropics compared to monocropping systems like maize plantations. [ABSTRACT FROM AUTHOR]

Knowledge on enzymatic mechanisms of acclimation to temperature is required to predict the effects of warming on decomposition of soil organic matter – the largest C stock in terrestrial ecosystems. Based on Michaelis–Menten kinetics we tested the hypothesis that enzyme affinity to substrate ( K m ) is more sensitive to warming at cold than at warm temperatures. We also predicted a gradual increase in K m values with increasing temperature. The kinetic parameters of six enzymes involved in cycles of C, (cellobiohydrolase, β-glucosidase and xylanase), P (phosphatase), and N (leucine-aminopeptidase, tyrosine-aminopeptidase) were determined after one month of soil incubation at a temperature range 0–40 °C (with 5° increment). Contrary to our hypothesis, the increase in K m with temperature was not gradual for most tested enzymes. Within large range of temperatures from 0 to 15 °C (phosphatase), 0–20 °C (enzymes involved in C cycle) and 0–40 °C (proteases) the hydrolytic activity was governed by enzymes with nearly constant substrate affinity. Temperature, therefore, mainly selected for soil enzyme systems maintaining static K m . The catalytic efficiency of the enzymes ( V max / K m ) increased from low to intermediate temperatures (0–20 °C) as a result of linear increase of V max at constant K m . Static K m values were explained either by low flexibility (high structural stability) of a single enzyme type, which catalyzed the reaction over a broad temperature range, or by production of multiple isoenzymes each with different temperature optima but with similar affinity to substrate. Thus, maintaining static K m with temperature increase ensured high enzyme efficiency within a low and intermediate soil temperature range. [ABSTRACT FROM AUTHOR]

The rhizosphere, the small soil volume that surrounds and is influenced by plant roots, is one of the most dynamic biological interfaces on Earth. Enzymes, produced by both roots and microorganisms, are the main biological drivers of SOM decomposition. In situ soil zymography was applied to test hypotheses that 1) the spatial pattern of rhizosphere activity is enzyme-specific and 2) the distribution of enzyme activity along the roots is dependent on root system and plant species. Lentil ( Lens culinaris ) and maize ( Zea mays L.), two species with contrasting root physiology, were chosen to test their effects on spatial distribution of activities of β-glucosidase, cellobiohydrolase, leucine-aminopeptidase and phosphatase. The extent of the rhizosphere for each enzyme and plant species was estimated as a function of distance from the root. For the first time, we demonstrated plant-specific patterns of exoenzyme distribution: these were uniform along the lentil roots, whereas in the rhizosphere of maize, the enzyme activities were higher at the apical or proximal root parts. We conclude that the shape and extent of the rhizosphere for enzyme activities is plant species specific and varies due to different rhizosphere processes (e.g. root exudation) and functions (e.g. nutrient mobilization abilities). The extension of enzyme activity into the rhizosphere soil was minimal (1 mm) for enzymes responsible for the C cycle and maximal (3.5 mm) for enzymes of the phosphorus cycle. This should be considered in assessments and modeling of rhizosphere extension and the corresponding effects on soil properties and functions. [ABSTRACT FROM AUTHOR]

Microbial and enzyme activities can be used to identify and assess the impacts of changes in land use management on soil quality. However, only few studies have investigated the effects of land use and nutrient additions on enzyme activities and microbial processes in tropical African soils. Glucose and nutrients (N and P) were added to soils (0–20 cm) from natural and agricultural ecosystems: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands (6) Chagga homegardens common at Mt. Kilimanjaro region and East Africa. Microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase were monitored over 60 days incubation period. Microbial biomass content and enzyme activities were generally higher in soils under natural vegetation compared to corresponding agricultural soils. Decline in microbial biomass C content over time was higher in natural ecosystems compared to agricultural soils. However, the microbial biomass C content in Chagga homegarden soils was relatively stable. Land use was negatively correlated to β-glucosidase, cellobiohydrolase and chitinase activity, but positively correlated to phosphatase activity. β-glucosidase and cellobiohydrolase, involved in the C-cycle, were the most sensitive to landuse change. Chitinase activity was 2–6 times higher in soils under natural vegetation compared to corresponding arable soils. Phosphatase displayed very high activities in all land use types. This is attributed to the high P retention capacity common for andic soils similar to those occurring at Mt. Kilimanjaro region. Increased P availability stimulated enzyme activities in lower montane forest and Chagga homegarden soils. Overall, microbial biomass and enzyme activities showed a strong decrease with increased land use intensity and should therefore be taken into consideration in monitoring and assessing the impact of land use change at Mt. Kilimanjaro region. [ABSTRACT FROM AUTHOR]

Paddies contain 78% higher organic carbon (C) stocks than adjacent upland soils, and iron (Fe) plaque formation on rice roots is one of the mechanisms that traps C. The process sequence, extent and global relevance of this C stabilization mechanism under oxic/anoxic conditions remains unclear. We quantified and localized the contribution of Fe plaque to organic matter stabilization in a microoxic area (rice rhizosphere) and evaluated roles of this C trap for global C sequestration in paddy soils. Visualization and localization of pH by imaging with planar optodes, enzyme activities by zymography, and root exudation by 14C imaging, as well as upscale modeling enabled linkage of three groups of rhizosphere processes that are responsible for C stabilization from the micro‐ (root) to the macro‐ (ecosystem) levels. The 14C activity in soil (reflecting stabilization of rhizodeposits) with Fe2+ addition was 1.4–1.5 times higher than that in the control and phosphate addition soils. Perfect co‐localization of the hotspots of β‐glucosidase activity (by zymography) with root exudation (14C) showed that labile C and high enzyme activities were localized within Fe plaques. Fe2+ addition to soil and its microbial oxidation to Fe3+ by radial oxygen release from rice roots increased Fe plaque (Fe3+) formation by 1.7–2.5 times. The C amounts trapped by Fe plaque increased by 1.1 times after Fe2+ addition. Therefore, Fe plaque formed from amorphous and complex Fe (oxyhydr)oxides on the root surface act as a "rusty sink" for organic matter. Considering the area of coverage of paddy soils globally, upscaling by model revealed the radial oxygen loss from roots and bacterial Fe oxidation may trap up to 130 Mg C in Fe plaques per rice season. This represents an important annual surplus of new and stable C to the existing C pool under long‐term rice cropping. [ABSTRACT FROM AUTHOR]

Biochar (BC) and nitrogen (N) fertilizers are frequently applied to improve soil properties and increase crop productivity. Nonetheless, our mechanistic understanding of plant-soil interactions under single or combined application of BC and N remains incomplete. For the first time, we applied a split-root system to evaluate how BC or N contributes to the changes in soil enzyme activities, N and phosphorus (P) cycling as well as root plasticity. Left and right parts of rhizoboxes were filled with silty-clay loamy soil amended with BC (15 g kg−1 soil, from wheat straw, 300 °C), N (0.05 g KNO 3 -N kg−1 soil) or a control (no amendments), resulting in the following combinations: BC/Control, N/Control, BC/N. Soil enzyme activities, available N and P, root morphology and plant biomass were analyzed after plant harvest. Plant biomass (shoot + root) ranged from 0.56 g pot−1 (BC/Control) to 0.91 g pot−1(BC/N). The decreased soil bulk density and increased P availability in the BC compartment (BC/Control and BC/N) stimulated root length by 1.4–1.8 times – an effect that was independent of N availability in the same rhizobox. Biochar stimulated activities of β-glucosidase and leucine aminopeptidase (by 33–39%) compared to N due to the coupling of C, N and P cycles in BC/N treated soil. Nitrogen fertilization also increased β-glucosidase activity compared to the unfertilized control, whereas root elongation remained unaffected. Thus, the combined application of BC/N had more efficient benefits for plant growth than BC or N alone. This is linked with i) the stimulation of enzyme activities at the BC locations to reduce N limitation for both microorganisms and plants, and ii) an increase of fine root production to improve N uptake efficiency. Thus, combined BC/N application is potentially especially sustainable to overcome nutrient limitation as well as to maintain crop productivity because it accelerates root-microbial interactions. Three-pairs design (BC/Control, N/Control and BC/N) in the rhizoboxes and main parameters (fine root length, enzyme activities and nutrients) related to plant growth strategy. Unlabelled Image • Split-root experiment was done to study the effect of fertilizers on maize growth. • Biochar (BC) and nitrogen (N) combination stimulated the highest growth of maize. • BC increased activities of β-glucosidase and leucine aminopeptidase in soil. • BC stimulated the root elongation of maize, whereas N had no effect. [ABSTRACT FROM AUTHOR]

The input of labile organics by plant roots stimulates microbial activity and therefore facilitates biochemical process rates in the rhizosphere compared to bulk soil, forming microbial hotspots. However, the extent to which the functional properties of soil microorganisms are different in the hotspots formed in soils with contrasting fertility remains unclear. We identified the hotspots related to different levels of Zea mays L. root architecture by zymography of leucine aminopeptidase in two soils with contrasting fertility. The hotspots localized by tiny wet-needle approach around first- and second-order roots were compared for parameters of microbial growth and enzyme kinetics. The pattern of hotspot distribution was more dispersed and the hotspot area was one order of magnitude smaller around first-versus second-order roots. The specific microbial growth rate (μ m) and biomass of active microorganisms were soil-specific, with no difference between the hotspots and bulk soil in the fertile soil. In contrast, in the soil poor in organic matter and nutrients, 1.2-fold higher μ m and greater growing biomass were found in the hotspots versus bulk soil. Lower enzyme affinity (1.3–2.2 times higher K m value) of β-glucosidase and leucine aminopeptidase to the substrate was detected in the hotspots versus bulk soil, whereas only β-glucosidase showed higher potential enzyme activity (V max) in the hotspots, being 1.7–2.1 times greater than that in bulk soil. Notably, the activity of C-acquiring enzyme, β-glucosidase positively correlated with the biomass of actively growing microorganisms. The fertile soil, on the whole, showed greater V max and catalytic efficiency (V max / K m) and an approximately 2.5 times shorter substrate turnover time as compared to the poor soil. Therefore, we conclude that i) the differences in microbial growth strategy between rhizosphere hotspots and bulk soil were dependent on soil fertility; ii) affinity of hydrolytic enzyme systems to substrate was mainly modulated by plant, whereas potential enzymatic activity was driven by both plant and soil quality. • The hotspots around roots were successfully localized by tiny wet-needle approach. • Stimulation on microbial growth in the hotspots was soil-specific. • Roots modulated enzyme affinity to substrate. • The fertile soil showed greater catalytic efficiency than the poor soil. [ABSTRACT FROM AUTHOR]

Legume–rhizobium symbiosis plays an important role in agriculture and ecological restoration. However, the regulatory mechanisms of rhizobium in alleviating heavy metal stress through the biochemical response of plant-soil system is limited. In this study, alfalfa was inoculated with a copper (Cu)-resistant rhizobium, and its effect on plant growth and the spatial distribution of phosphatase in the rhizosphere under Cu stress was assessed. Our results showed that rhizobium inoculation alleviated Cu-induced growth inhibition, and increased the nitrogen and phosphorus content in alfalfa tissues. Moreover, inoculated plants had a higher Cu uptake than non-inoculated plants, with a much higher increase in the roots than in the shoots; thus, inoculation with rhizobium was shown to decrease the transfer coefficient and promote Cu phytostabilization. The zymograms illustrated that the distribution of phosphatase activities was associated with the presence of roots. Compared with the non-inoculated treatment, the rhizobium inoculation increased the hotspot areas of phosphatase by 26.1% and 39.3% at the Cu 0 and Cu 800 treatments, respectively. In addition, the available phosphorus in the soil showed negative correlations with soil phosphatase activity (p < 0.05). The model of partial least squares path modelling (PLS-PM) indicated that soil Cu content directly influenced the hotspot areas of phosphatase activities in the rhizosphere and explained most (86%) of the variation. Thus, the enzymatic hotspots were concluded to mainly be affected by the Cu content of soil, and the phosphatase activities in the rhizosphere were mainly regulated by the ratio of nitrogen to phosphorus. These findings provide a basis for the spatio-temporal dynamics of biogeochemical reactions in the rhizosphere of polluted soils. Image 1 • The distribution of phosphatase activity was associated with the presence of roots. • Rhizobium increased phosphatase activity in the high Cu-polluted soil. • The enzymatic hotspot is primarily affected by the Cu content of soil. • The phosphatase activity mainly regulated by the ratio of nitrogen to phosphorus. [ABSTRACT FROM AUTHOR]

Successive winter wheat (WW) rotations are associated with a substantial yield decline, and the underlying mechanisms remain elusive. An outdoor experiment was set up using sandy loam soil. WW was grown in rhizotrons, in soil after oilseed rape (KW1), after one season of WW (KW2), and after three successive seasons of WW (KW4). We applied zymography and harvested the plants at the stem elongation stage to observe changes in the activity of β -glucosidase (BGU) and leucine aminopeptidase (LAP), as well as using glucose (GLU) imaging to observe glucose release patterns in the rhizosphere of WW. Several biochemical and microbial properties of the bulk soil and the rhizosphere of the rotational positions were measured. KW2 and KW4 exhibited reduced plant biomass compared to KW1. There was a higher root length density and root mean diameter as well as a lower specific root length for KW1 compared to KW2 and KW4. KW1 soil had a lower mineral N concentration and microbial biomass carbon (C) and nitrogen (N) than KW2 and KW4, which translated to a lower plant C:N ratio. A greater rhizosphere extent of BGU and LAP across the soil profile was also visible for KW1 compared to KW2 and KW4 using zymography. Lower dissolved organic C and hotspot areas of GLU in the rhizosphere of successive WW might explain shifts in the microbial community composition, possibly leading to a dysbiosis with the soil microbes in the rhizosphere. Soil depth and rotational position explained most of the variance in the soil microbial communities. The relative abundance of Acidobacteriota , Gemmatimonadota , Nitrospirota , and Chloroflexi significantly varied among the rotational positions. Our results highlight the effect of WW rotational positions on soil and plant properties, as well as microbial community dynamics, and provide evidence for the pathways driving biomass decline in successively grown WW. [Display omitted] • Significant biomass decline in successively grown winter wheat (WW). • Contrasting root plastic responses in response to successive WW rotational positions. • Lower DOC in the rhizosphere of successively grown WW was related to microbial N immobilization. • WW rotational position strongly affected activity of C and N- acquiring enzymes. • Microbial community composition is affected by WW rotational position. [ABSTRACT FROM AUTHOR]

Roots are ecosystem engineers and build their environment, shaping the rhizosphere for optimal functioning. The interactions between plants and soil microbes are an important focus of terrestrial ecology. The application of classical and novel in situ imaging approach in combination with stable and radioactive isotopes to trace C and N fluxes shade light on the crucial role of root exudates on nutrient acquisition in the rhizosphere depending on biotic (e.g. pathogen, plant diversity) and abiotic factors (e.g. drought, temperature and land use). High-quality and quantitative images clearly showed that extend of rhizosphere is plant specific and it depends on root morphology; nutrient limitation and pathogenic infection will enlarge the rhizosphere size and increase soil volume from which root can mine nutrient. Overlapping of zymography and 14C imaging combined with qPCR analysis showed that thirsty maize release more root exudates and stimulates microbiome to express more peptidases. Similarly, coupling imaging and analytical tools illustrate while acclimation of enzymes to temperature showed that warming, mainly selected for soil enzyme systems maintaining static substrate affinity, it can also shorten the duration of hotmoment and increased the hotspots. Similarly, land use management strategies and fertilization revealed evidence of 1) changes in enzyme systems or 2) shift in microbial community and 3) spatial (re)distribution of enzymatic hotspots. In addition, this methodological combination showed that the rhizosphere shape for all parameters corresponds to sigmoidal or diffusion curves. Sharp gradients are formed within a few days for nutrients and enzymes. Showing, despite the dynamic nature of each property, rhizosphere shape is quasi-stationary because of the opposite directions of its formation processes. Thus, smart conjugation of classical and new approaches for static imaging and dynamic visualization of soil environments will benefit soil (micro)biology by providing new insights of life in the opaque belowground. Detecting and locating (micro)organisms poised soil (micro)biology to progress from simply cataloguing organism complexity to becoming a functional system science. [ABSTRACT FROM AUTHOR]

• Warming facilitates the consumption of labile organics because of the faster microbial growth. • Microbial respiration and enzyme activities acclimates to 8-year warming. • The increased labile organics weakens the memory effect of soil microbial functions. • Microbial memory to warming is strongly dependent on microbial physiological state. The feedback between soil carbon (C) and climate change has the potential to decrease over time, which is mainly due to the thermal acclimation of microbial decomposition of soil organic matter (SOM). The adaption and stabilization mechanisms of microbial functions in response to long-term warming in agroecosystems, however, remain intensive debate. Therefore, we explored the controls of thermal acclimation based on an 8-year field warming (ambient, +1.6 °C, +3.2 °C) in agroecosystem and a short-term incubation at five constant temperature levels (from 5 to 25 °C with 5 °C intervals) under microbial steady-state (without glucose, non-activated microbial community) and activate microbial growth induced by sufficient glucose addition. At the steady-state, field warming (+3.2 °C) increased the specific growth rate whilst decreased the portion of growing microbial biomass as compared to ambient soils. This indicated that 8-year field warming facilitated the consumption of labile organics due to faster microbial growth. Consequently, microbial growth, activities of β-glucosidase and leucine aminopeptidase, as well as microbial respiration acclimated to field warming (+1.6 °C, +3.2 °C) over 8-year, responded up to 40% weaker to further short-term temperature increase (above 15 °C). Also, the temperature sensitivity of enzyme activity decreased with field warming magnitude under higher incubation temperatures, which further indicated enzyme acclimation. Under activation mode, however, the higher enzyme activities suggested that the increased labile C remarkably relieved the substrate limitation of microbial growth and activated dormant microorganisms in field warmed soils, and weaken the thermal acclimation of soil microbial functions. We thus found experimental evidence that the transition between microbial physiological states (i.e., dormant vs. active) owing to variations in C availability is the most plausible explanation for the alterations in temperature sensitivity of enzyme activities and microbial respiration. The compensating effect of the increased labile substrate under climate warming on the thermal acclimation of soil microbe-driven CO 2 may thus be larger than that currently predicted, with important consequences for atmospheric CO 2 concentrations. [ABSTRACT FROM AUTHOR]

Soil zymography is a new technique developed to visualize two‐dimensional distributions of enzyme activities. The method consists of incubating a membrane saturated with an enzyme‐specific fluorogenic substrate on a surface of the soil sample, followed by recording the membrane image generated by a fluorescent product (e.g. MUF: methylumbelliferone) in ultraviolet light. Despite its relative ease of use, performing zymography involves multiple user‐made decisions that might affect the accuracy of enzyme activity estimates. Therefore, unification of the zymography methodology is required for correct estimations and comparisons of various studies. We evaluated the following methodological aspects of the implementation of zymography: (a) camera settings and image processing, (b) effects of evaporation and (c) calibration procedures. Camera settings (shutter speeds or exposure time) affected the intensity of background fluorescence and signal‐to‐noise ratios (SNR). However, because their combined effects varied depending on MUF concentrations, light and camera setting need to be optimized for the expected range of MUF concentrations prior to zymography. Evaporation of MUF solution from the membrane had no effect on fluorescence. Relations between MUF concentration and intensity of fluorescence during calibrations demonstrated a saturated pattern and were strongly affected by image noise outside the optimal range (e.g. 8–14 μm MUF pixel−1). We developed a new calibration approach that is based on a piecewise linear regression. The new approach accounted for specific ranges of MUF concentration and uses nonuniformly saturated membranes, reflecting the real distribution of enzyme activities in soil. The new calibration algorithm eliminated biases of the standard calibration and resulted in greater accuracy in predicting MUF concentrations. Highlights: We developed a new approach to calibration for 2‐D soil zymography.The approach accounted for spatial nonuniformity of soil zymograms.Standard calibration resulted in systematic underestimation of enzyme activity.Soil zymography requires pixel‐based calibration with nonuniformly saturated membranes. [ABSTRACT FROM AUTHOR]

Abstract As a homologous gene encoding microbial alkaline phosphomonoesterase, the expression of phoD is critically controlled by P availability and thus contributes to the mineralization of soil organic P under P-depleted condition. However, its role in the regulation of soil P turnover is largely unknown due to the complex coupling of physiochemical and biological processes in the P cycle, especially in paddy field. We hypothesized that 1) P fertilization would decrease the abundance of phoD gene and change the composition of phoD -harboring microbial community and 2) the high abundance of phoD -harboring microorganisms in P-poor soil would stimulate the synthesis of alkaline phosphomonoesterase, thus mitigating P limitation via the mineralization of organic P. After 42 days of rice growth, the phoD abundance negatively correlated with soil P availability, and it was significantly higher in non-fertilized treatments than in P-fertilized treatments for both rhizosphere and bulk soils. A stronger competition among phoD -harboring microorganisms was detected in non-fertilized soil than in P-fertilized soil, with Bradyrhizobium , Methylobacterium, and Methylomonas being the dominant taxa in all samples. However, the high phoD gene abundance under P-poor condition was mainly due to the growth of rare operational taxonomic units (OTUs) affiliated to Actinobacteria and Cyanobacteria (relative abundance < 3%). Consistent with our hypothesis, the growth of phoD -harboring microorganisms stimulated the hydrolysis of organic P in non-fertilized soil. However, in the P-fertilized treatments, the increase in OTU abundance was accompanied by the depletion of exchangeable P and accumulation of microbial biomass P. Our findings suggest that phoD -harboring microorganisms have the potential to immobilize P in biomass when the supply is sufficient while mineralize organic P under P-poor condition, during which the rare taxa play an important role. Graphical abstract Image 1 Highlights • Competition among phoD -harboring microbes is intense under P-poor conditions. • Increase in phoD gene abundance is related to the growth of rare species. • phoD -harboring microbes store P in biomass under P-sufficient condition. • phoD -harboring microbes might reuse the intracellular P when P is depleted. [ABSTRACT FROM AUTHOR]

Abstract Soil membrane zymography enables 2D mapping of enzyme activities on the surface of soil samples. The method is based on diffusion of components of enzymatically-mediated reactions to/from membrane, and, thus, reflects the distribution of enzyme activities at the intact soil surface. Zymography has been already successfully implemented in numerous soil ecology applications. Here we identify two methodological aspects for further improvement and expansion of the method at micro and macro scales: first, accounting for the area of contact between the soil surface and the zymography membranes and, second, accounting for diffusion effects during the zymography procedure. We tested three methods, namely, laser-scanning, staining with a fluorescent product (e.g. MUF: 4-methylumbelliferone), and X-ray computed micro-tomography, for assessing the area of the soil surface in contact with the membranes. We quantified diffusion of MUF, enzymes and substrate between the substrate-saturated membrane and soil as well as diffusion processes during membrane zymography via HP2 software. Diffusion of the substrate from the membrane and of the MUF-product to the membrane was detected, while there were no clear evidence of enzyme diffusion to/in the membrane. According to the model simulations, the enzyme activities detected via 2D zymography probably represent only a small portion, about 20%, of the actual reactions within the soil volume that is in both direct contact and in hydrological contact with zymography membranes. This is a result of omnidirectional diffusion of reaction products. The membrane contact with the soil surface estimated by three methods ranged from 3.4 to 36.5% further signifying that only a fraction of enzymes activity is detectable in a course of 2D soil zymography. Highlights • We studied processes occurring in the soil and the membrane during 2D soil zymography. • We modeled product and substrate diffusion in the soil/membrane. • Zymography underestimates enzyme activity due to product diffusion losses in soil. • Interpretation of 2D zymography requires measurements to be combined with modeling. [ABSTRACT FROM AUTHOR]

Deadwood is a key component of forest ecosystems, but there is limited information on how it influences forest soils. Moreover, studies on the effect of thinning-derived deadwood logs on forest soil properties are lacking. This study aimed to investigate the impact of thinning-derived deadwood logs on the soil chemical and microbial properties of a managed spruce forest on a loamy sand Podzol in Bavaria, Germany, after about 15 years. Deadwood increased the soil organic carbon contents by 59% and 56% at 0–4 cm and 8–12 cm depths, respectively. Under deadwood, the soil dissolved organic carbon and carbon to nitrogen ratio increased by 66% and 15% at 0–4 cm depth and by 55% and 28% at 8–12 cm depth, respectively. Deadwood also induced 71% and 92% higher microbial biomass carbon, 106% and 125% higher microbial biomass nitrogen, and 136% and 44% higher β-glucosidase activity in the soil at 0–4 cm and 8–12 cm depths, respectively. Many of the measured variables significantly correlated with soil organic carbon suggesting that deadwood modified the soil biochemical processes by altering soil carbon storage. Our results indicate the potential of thinned spruce deadwood logs to sequester carbon and improve the fertility of Podzol soils. This could be associated with the slow decay rate of spruce deadwood logs and low biological activity of Podzols that promote the accumulation of soil carbon. We propose that leaving thinning-derived deadwood on the forest floor can support soil and forest sustainability as well as carbon sequestration. [ABSTRACT FROM AUTHOR]

Summary: Root hairs and soil water content are crucial in controlling the release and diffusion of root exudates and shaping profiles of biochemical properties in the rhizosphere. But whether root hairs can offset the negative impacts of drought on microbial activity remains unknown.Soil zymography, 14C imaging and neutron radiography were combined to identify how root hairs and soil moisture affect rhizosphere biochemical properties. To achieve this, we cultivated two maize genotypes (wild‐type and root‐hair‐defective rth3 mutant) under ambient and drought conditions.Root hairs and optimal soil moisture increased hotspot area, rhizosphere extent and kinetic parameters (Vmax and Km) of β‐glucosidase activities. Drought enlarged the rhizosphere extent of root exudates and water content. Colocalization analysis showed that enzymatic hotspots were more colocalized with root exudate hotspots under optimal moisture, whereas they showed higher dependency on water hotspots when soil water and carbon were scarce.We conclude that root hairs are essential in adapting rhizosphere properties under drought to maintain plant nutrition when a continuous mass flow of water transporting nutrients to the root is interrupted. In the rhizosphere, soil water was more important than root exudates for hydrolytic enzyme activities under water and carbon colimitation. See also the Commentary on this article by Zhang et al., 237: 707–709. [ABSTRACT FROM AUTHOR]

The influence of grazing on soil seed bank (SSB) characteristics determines the management of rangelands in different climates. Here we studied the responses of SSBs to livestock grazing in three regions with different climates – arid, semiarid and subhumid – in northern Iran. In each region 30 pairwise SSB samples were collected on 1 m2 plots, 15 in intensely grazed areas and 15 in ungrazed areas. The total SSB densities, their diversity and species richness and the densities of functional groups were compared between the grazed and ungrazed plots and between the climatic regions. Both climate and grazing as well as their interaction affected SSB characteristics. Although the results of the non-metric multidimensional scaling (NMDS) showed noticeable differences in SSB species composition between grazed and ungrazed plots in all three regions, the magnitude of the grazing effect on the SSB was different between climatic regions. In total, 119 species germinated from the soil samples: 33 species in semiarid, 39 species in arid and 61 species in subhumid samples. The highest total SSB density, diversity (Shannon index) and richness were found in subhumid climate with an average of 138.90 seeds/m2, H = 2.27 and 17.83 species/sample, respectively. In ungrazed areas the average SSB density was 26.60 seeds/m2, 46.06 seeds/m2 and 195.90 seeds/m2 in arid, semiarid and subhumid climate, respectively. In grazed areas the corresponding figures were 12.40 seeds/m2, 7.00 seeds/m2 and 110.40 seeds/m2. Averages of SSB diversity in ungrazed areas were 1.24, 1.60 and 2.42 in arid, semiarid and subhumid climates, respectively, as compared to 1.27, 0.97 and 2.20 in grazed areas. The averages SSB richness in ungrazed areas were 5.21 species/sample, 7.54 species/sample and 21.25 species/sample in arid, semiarid and subhumid climate, respectively, whereas in grazed areas 4.00 species/sample, 2.86 species/sample and 16.15 species/sample were found on average. Overall, intensive grazing was linked to lower SSB characteristics in all three climatic regions, but the size of the effect differed between the climatic regions. Thus, we concluded that the impact of grazing on SSB density, diversity and richness is climate-dependent. Hence, the climatic conditions have to be considered when evaluating the effects of grazing on soil seed banks. • Soil seed bank (SSB) is an important part of plant communities. • We studied the responses of SSB to grazing in three regions with different climates. • Intensive grazing suppressed SSBs, consistently across all climatic regions. • The magnitude of impact of grazing on SSB characteristics is climate-independent. [ABSTRACT FROM AUTHOR]

Mulching is widely adopted in arid regions to improve soil environment and crop productivity. However, our understanding of the underlying mechanisms of such improvements from enzyme activity and rhizosphere microbial community structure perspectives remain poor. To fill the gap, soil zymography was coupled with high-throughput sequencing to investigate enzyme activities and bacterial community structure in a 10-year field experiment in a semi-arid region. We examined the effects of two mulching measures (plastic film mulching [FM] and straw mulching [SM], with no mulching as the control [CK]) on the maize rhizosphere extent of β-glucosidase and leucine aminopeptidase activities and the abundance of the bacterial genes encoding these two enzymes. The enzyme activity in the SM and FM measures was higher compared to the CK measures (P < 0.05). SM had higher influence than FM on β-glucosidase activity and its rhizosphere extent. This was related to the increase in soil organic carbon (SOC) content and the stable bacterial community structure and interactions under SM. In contrast, leucine aminopeptidase activity was the highest under FM due to the higher soil temperature and moisture content under FM which further intensified nutrient competition among microorganisms or between microorganisms and plants. In addition, Proteobacteria and Acidobacteria were the major contributors to β-glucosidase activity despite their genes encoding β-glucosidase not increasing under SM. Conversely, the copy number of genes encoding leucine aminopeptidase and the genes encoded by Proteobacteria (the main contributor) decreased under FM. Therefore, total gene number is not a stable indicator of microbial function (such as enzyme activity) and cannot explain enzyme activity adequately. Overall, this study establishes a connection between rhizosphere enzyme activity and bacterial communities, revealing the mechanisms by which changes in environmental nutrient supply and bacterial community composition under straw mulching and plastic film mulching measures lead to variations in enzyme efficiency. • SM had greater influence on β-glucosidase activity and its rhizosphere extent. • Higher β-glucosidase activity under SM linked to substrate availability. • FM had highest leucine aminopeptidase activity due to moisture and temperature. • Genes encoding β-glucosidase and leucine aminopeptidase showed opposite patterns. [ABSTRACT FROM AUTHOR]

Metal contamination from mine waste is a widespread threat to soil health. Understanding of the effects of toxic metals from mine waste on the spatial patterning of rhizosphere enzymes and the rhizosphere microbiome remains elusive. Using zymography and high-throughput sequencing, we conducted a mesocosm experiment with mine-contaminated soil, to compare the effects of different concentrations of toxic metals on exoenzyme kinetics, microbial communities, and maize growth. The negative effects of toxic metals exerted their effects largely on enzymatic hotspots in the rhizosphere zone, affecting both resistance and the area of hotspots. This study thus revealed the key importance of such hotspots in overall changes in soil enzymatic activity under metal toxicity. Statistical and functional guild analysis suggested that these enzymatic changes and associated microbial community changes were involved in the inhibition of maize growth. Keystone species of bacteria displayed negative correlations with toxic metals and positive correlations with the activity of enzymatic hotspots, suggesting a potential role. This study contributes to an emerging paradigm, that changes both in the activity of soil enzymes and soil biota – whether due to substrate addition or in this case toxicity – are largely confined to enzymatic hotspot areas. [Display omitted] • The interaction between enzyme activities and mine soil was analyzed through zymography. • Mine soil inhabited the growth of maize. • Mine soil decreased the area and resistance of enzyme hotspots. • The enzyme hotspots are highly related to bacterial keystone species and co-occurrence characteristics [ABSTRACT FROM AUTHOR]

The spatial distribution of alkaline phosphomonoesterase (ALP) activity in the rhizosphere and bulk soil of three plants (Zea mays, Medicago sativa, and Cyperus rotundus) grown in a karst soil (pH 8.27) was visualized using in situ soil zymography. According to the zymogram images, we identified and precisely collected soil samples from hotspots and non-hotspots of ALP activity, and then analyzed the phoD genes that encoded alkaline phosphomonoesterase and assessed the microbial community. The results showed that (1) the phoD abundance in the plant types varied and was highest in the alfalfa and lowest in the maize; (2) Proteobacteria dominated the phoD-harboring microbes in the rhizosphere and Actinobacteria dominated the phoD-harboring microbes in the bulk soil, and (3) the ALP activity was positively correlated with the relative abundance of the diazotrophic Azotobacter, but negatively correlated with the phoD gene abundance, microbial community richness, and diversity. By coupling zymography and microbial molecular approaches, we identified hotspots of enzymatic and microbial activity in rhizosphere soil and evaluated the relative contributions from potential active microorganisms. We found that the function of specific phoD-harboring microorganisms in these hotspots differed depending on the plant in the soil, which had implications for phosphorus (P) management in P-limited karst soils. The results also suggested that free-living N2-fixing bacteria (Azotobacter) might promote ALP activity, thereby emphasizing vital linkages between, and coupling of, the soil nutrient cycles. [ABSTRACT FROM AUTHOR]

Phosphorus (P) application is an indispensable practice in agricultural crop production. However, the effect of P on crop and microbial biomass stoichiometries as well as its role in soil nutrient dynamics, remain poorly understood, thereby limiting our ability to predict the ecological consequences of agricultural practices. Herein, we conducted a P application experiment in a subtropical P-limited paddy soil to examine the effect of P addition on soil nutrient availability as well as on rice and the microbial C:N:P stoichiometries. The results showed that soil dissolved organic carbon (DOC) and available P content were simultaneously affected by P application and rice growth stage. Further, addition of P served to increase rice C and N content while decreasing the C: P and N: P stoichiometric ratios, indicating that P input contributed to rice growth and N absorption. Marked increases in soil microbial biomass carbon (MBC) and phosphorus (MBP) were also observed in soil treated with P. Moreover, the microbial C:N:P stoichiometry increased with rice growth stage, suggesting that changes in microbial and plant nutrient demand throughout the rice plant growth cycle causes competition between microorganisms and plants for N and P. Pearson's correlation and redundancy analyses revealed that available nutrients of soil were intercorrelated and regulated the patterns of rice and microbial C:N:P stoichiometries. In conclusions, P enrichment altered soil nutrient composition, microbial nutrient mining strategies, and rice C:N:P stoichiometry, thereby contributing to rice growth and improving crop yield. • P addition increased rice C and N pool and decreased rice stoichiometric ratios of C: P and N: P. • P input induced C and N limitations in the soil and microbial biomass. • The soil microbial ecological C: N: P stoichiometry varied with the rice growth stage. • Altered soil C: N: P stoichiometr affect the P strategy and N fixation of plants. [ABSTRACT FROM AUTHOR]

Aims: The rhizosphere has ecological importance as a microbial hotspot to understand the 'real' processing rates of element cycles without being misled by inactive bulk soil. It is thus essential to estimate the rhizosphere size and its response to anthropogenic distribution during crop growth. Methods: In situ β-glucosidase, cellobiohydrolase (C-acquiring), and acid and alkaline phosphatase (P-acquiring) activity was examined using soil zymography in rice rhizosphere. Temporal dynamics of enzymatic rhizosphere size under phosphate and cellulose fertilization were calculated based on the expansion of enzyme activity hotspot. Results: After 35 days of root development, radial expansion of cellobiohydrolase and acid phosphatase from the root centre to bulk soil was further than that of β-glucosidase and alkaline phosphatase, respectively. Root development expanded β-glucosidase hotspot in rhizosphere, but inhibited rhizosphere expansion of phosphatase activities. P fertilization caused strong N competition between plants and microorganisms, reducing rhizosphere expansion of all tested enzyme activities. Cellulose had no significant effects on C-acquiring enzyme activity expansion, but led to extended acid and alkaline phosphatase hotspots in the rice rhizosphere under P fertilization. Conclusion: i) Plant growth stage affects the rice enzymatic rhizosphere size; ii) P fertilization in P-limited soil enhances rhizosphere enzyme activities but reduces the radial expansion; iii) non-labile C application affects enzymatic rhizosphere expansion in an enzyme-specific manner interactively with P fertilization. [ABSTRACT FROM AUTHOR]

Phosphorus (P) availability affects the spatial and temporal distribution of phosphatase activity and acidification in the rhizosphere: two strongly interactive key strategies of nutrient acquisition by roots. Zymography was coupled with pH planar optode mapping to reveal the effects of P-availability (P deficiency, or with phytate or Ca(H 2 PO 4) 2 amendment) on the spatial distribution of phosphatase activity, pH and rhizosphere extent around the taproot of lupine before and after cluster root formation. Before cluster root formation, phosphorus deficiency increased acid phosphatase activities by 20%, decreased pH by 0.8 units and broadened the rhizosphere extent by 0.4 mm across the taproot. Phytate (80 mg kg−1) addition dampened these changes before cluster root formations. In contrast, the rhizosphere extent of phosphatase activity after cluster root formation was 0.2 mm narrower under P-deficiency than with Ca(H 2 PO 4) 2 amendment. Due to cluster root formation, the hotspot areas of alkaline phosphatase activity were 40% larger for lupine grown under P-deficiency than amended with Ca(H 2 PO 4) 2. Lupine rhizosphere strategies shifted during growth: increasing phosphatase activity, acidifying soil and broadening the rhizosphere around the taproot are dominant mechanisms before cluster root formation. After cluster root development, the main mechanism is increasing the area of phosphatase activity hotspots around cluster roots to enlarge the exploited soil volume. Image 1 • P deficiency increased phosphatase activity, decreased pH across the taproot before cluster root formation. • Phytate amendment reduced phosphatase activity and soil acidification. • PH increased and its rhizosphere extent narrowed around taproot after cluster root development. • Phosphatase activity hotspots area increased after cluster root development under P deficiency. [ABSTRACT FROM AUTHOR]

[Display omitted] • Enzyme activities decreased during prolonged drought but partly or totally recovered after rewetting. • Reallocation of resources by microbes as a response strategy to dry-rewetting (D/W). • MBC positively correlated with chitinase and β-glucosidase activities at the onset of drought. • 60% WHC rewetting influenced enzyme systems while 100% WHC flooding induced enzyme synthesis. How environmental history mediates the response of microbial activities to dry-wet cycle (D/W) in monsoon regions is poorly investigated. In this study, soil from Red River Delta of Vietnam was incubated for 52 days, which included 14 days at 20% water holding capacity (WHC), followed by 14 days rewetted to 60% WHC (rewetting) or 100% WHC (flooding). The kinetics of β-glucosidase, chitinase and acid phosphomonoesterase enzymes, microbial respiration, microbial biomass C and P were measured. The activities of all soil enzymes (V max) decreased by 20–42% due to drought but partially or totally recovered after two weeks of rewetting or flooding. The substrate affinity (K m) of all measured enzymes were fluctuating at initial drought and then dropped down by 33–55% after 14 days prolonged drought. Chitinase was the only enzyme showing the decoupling of V max and K m at the onset of drought, indicating different factors regulating enzyme synthesis and substrate affinity for the N demands of microorganisms. Pulse rewetting significantly reduced catalytic efficiency (K a) but boosted turnover time (T t) of chitinase and β-glucosidase. Rewetting prolonged T t as compared to flooding by the end of the experiment. Moreover, both soil basal respiration and substrate induced respiration were significantly inhibited by drought and recovered to the control level after rewetting. In conclusion, soil with history of D/W triggers the recovery of microbial activities after D/W cycles and rewetting has a great impact on enzyme systems, while flooding induces more effects on enzyme synthesis. [ABSTRACT FROM AUTHOR]

Enzymes are sensitive indicators of changes in soil, expressing the interaction between plant roots and microorganisms especially within rhizosphere. However, the C-rich rhizosphere also attracts and accommodates pathogens, such as root-knot nematode (M. incognita). The direct effects of nematodes on above and belowground plant parts are well known, but the broad range of indirect effects, especially on carbon (C) and phosphorus (P) cycles underground, remains unknown. Enzyme visualization was acquired in this study to tackle two hypotheses: i) cellobihydrolase activity will be lower after nematode infestation due to more labile C (sugars) in the rhizosphere; ii) plant roots produce more P-acquiring enzymes in response to their P stress, caused by nematodes, leading to a larger spatial extension of phosphatase than cellobiohydrolase. Eight lupine (Lupinus polyphyllus L.) plants were grown in separated rhizoboxes, four of which were inoculated with aqueous suspension containing 50 Meloidogyne incognita. Enzyme activities of cellobiohydrolase and acid phosphatase were visualized using membranes saturated with 4-methylumbelliferone (MUF) substrates. Plant resistance and resilience to nematode infestation by both modifying their root morphology and altering rhizosphere enzyme extent. However, enzymatic response to nematode effect is enzyme specific, while hotspot area of cellobiohydrolase was 20 times lower, acid phosphatase was 6 times higher in infected plants. Most importantly, for increasing P demand due to nematode attack, the plants enhanced phosphatase production in their lateral roots. Thus, the infested roots, rather than microorganisms, play a pivotal role in producing phosphatase. Importantly, this 1 mm increment of rhizosphere extent in 2D equals a 2-fold increase in soil volume (3D) for nutrient mobilization. In conclusion, using zymography to map the footprint of nematodes in the soil, we conclude that nematode infection not only has direct effects by changing root morphology, but also induces a number of subsequent biochemical changes in the rhizosphere, affecting C and P cycling. [ABSTRACT FROM AUTHOR]

Abstract Roots increase microbial activities depending on exudate composition, especially on the ratios of sugars, carboxylic and amino acids, and thus structure enzyme activities in the rhizosphere. We introduce a new approach combining soil zymography and simulated exudates released from Rhizon® samplers to stimulate microbial activities but avoid the direct release of enzymes by living roots. This enabled visualizing, localizing and analyzing the effects of simulated root exudates on activity of five microbial enzymes involved in carbon (C) (β-glucosidase, cellobiohydrolase), nitrogen (N) (leucine aminopeptidase), phosphorus (P) (phosphatase) and sulfur (S) (sulfatase) cycles. We tested the hypotheses that 1) artificial exudates stimulate microorganisms for enzyme production and form spatial gradients around roots, and 2) the extent of microbial enzyme activities in the rhizosphere is component-specific. In line with these hypotheses, the activities of P-, N- and S-related enzymes were higher near the artificial root and gradually decreased as a function of distance from the root. The pattern for C-cycle enzymes was uniform and independent of the exudate composition. Among all components, alanine increased the rhizosphere extent much stronger than other substances, while methionine had no effect on the spatial distribution of enzyme activities. V max of all enzymes increased with alanine addition, but decreased after adding citrate. The ratios of enzyme activities demonstrated that rhizosphere microorganisms release more leucine aminopeptidase than other enzymes to meet their N demand. Glucose increased the K m of cellobiohydrolase and β-glucosidase, while alanine had the greatest effect on the K m of leucine aminopeptidase and sulfatase. Phosphatase is the enzyme most sensitive to the composition of root exudates; consequently, any factor influencing root exudate composition can strongly affect the P cycle. We conclude that the rhizosphere extent of microbial-derived enzyme activities is component- and enzyme-specific and that this extent depends on the substrate stoichiometry and microbial nutrient demand. Graphical abstract Image 1 Highlights • We introduce a new approach to avoid direct release of enzymes by roots. • Distributions of microbial enzyme activities were enzyme- and component-specific. • Alanine increased the rhizosphere extent stronger than other substances. • Phosphatase had the most sensitive enzyme system to exudate composition. [ABSTRACT FROM AUTHOR]

Lentil and lupine, having contrasting root morphologies, were chosen to investigate the effects of plant growth and root morphology on the spatial distribution of β-glucosidase, cellobiohydrolase, leucine aminopeptidase, and acid phosphomonoesterase activities. Lentil kept as vegetative growth and the rhizosphere extent was constant, while the enzyme activities at the root surface kept increasing. Lupine entered reproductive growth in the seventh week after planting, the rhizosphere extent was broader in the eighth week than in the first and fourth weeks. However, enzyme activity at the root surface of lupine decreased by 10-50% in comparison to the preceding vegetative stage (first and fourth weeks). Lupine lateral roots accounted for 1.5-3.5 times more rhizosphere volume per root length than taproots, with 6-14-fold higher enzyme activity per root surface area. Therefore, we conclude that plant growth and root morphology influenced enzyme activity and shape the rhizosphere as follows: the enzyme activity in the rhizosphere increased with plant growth until reproductive stage; lateral roots have much larger rhizosphere volume per unit root length and higher enzyme activity per root surface area than the taproots. [ABSTRACT FROM AUTHOR]

The rhizosphere and detritusphere are hotspots of soil enzyme-mediated microbial processes, but little is known about their spatio-temporal distribution and interactions in situ , especially after straw incorporation. To answer this question, we planted maize in rhizoboxes with three wheat straw localizations: a) straw incorporated in the upper 2 cm of soil (straw localized); b) straw mixed with all the soil (straw homogenized); and 3) without straw (control). Zymography was used to investigate the spatio-temporal distribution of soil C- and N- acquiring enzyme activities in the rhizosphere (living roots) and detritusphere (straw induced), which depends on straw localization (localized vs. homogenized) and time (7 vs. 15 days after planting). The hotspot area of enzyme activities was similar in the detritusphere compared to the rhizosphere at the early stage (day 7), suggesting the importance of detritusphere in driving C and N cycling. Compared with homogenized straw, higher C- and N-acquiring enzyme activities in detritusphere were observed in the treatment with localized straw, which indicates that straw localization had a marked impact on the enzyme activity in the detritusphere. Although enzyme activities in rhizosphere and detritusphere decreased with time from straw application, straw incorporation increased the enzyme activities in the rhizosphere (especially with localized straw) compared to control at the late stage (day 15). Furthermore, homogenized straw greatly reduced plant height (−36 %), leaf area (−49 %), SPAD (−49 %), and shoot weight (−53 %) than control at the late stage, but localized straw had little impact on plant performance. In conclusion, straw incorporation forms another soil enzyme activity hotspot (detritusphere) and promotes enzyme activities in the rhizosphere and soil profile. Collectively, homogenized straw incorporation intensified nutrient competition between microorganisms and roots and restricted plant performance. [Display omitted] • The hotspot area of enzyme activity was similar in rhizosphere and detritusphere. • Enzyme activities in rhizosphere and detritusphere decreased over time. • Straw incorporation increased the enzyme activity in rhizosphere and soil surface. • Straw homogenized inhibited plant growth as nutrient competition between roots and microbes. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) are ubiquitous in the environment and can potentially damage microbes and plant root cells. Although the influence of MPs on soil parameters has been investigated, the response of microbiomes to soil microenvironments with contrasting limiting factors, particularly in flooded soil environments such as rice paddies, remains unknown. Using zymography and high-throughput sequencing, we conducted a mesocosm experiment with polylactide (PLA) and polyvinyl chloride (PVC) MPs to compare the effects of biodegradable and conventional MPs on rice growth, exoenzyme kinetics, and microbial communities. Both conventional and biodegradable MPs significantly inhibited rice growth, possibly by affecting nutrition. Compared with the control soils, both PLA- and PVC-amended soils exhibited higher enzyme activity in the hotspots. The enzymatic resistance to MPs was higher in 'coldpots' with PVC addition compared to that in PLA and control treatments. Bacterial biomass increased but diversity declined in PLA-amended soils, possibly because PLA particles act as carbon input inhabited the population of bacteria. Our findings suggest that co-occurrence networks among bacteria were strengthened by the addition of both MPs, with an increase in microbial functionality resilience and enhanced competition with neighboring roots for nutrient mining. This competition for nutrients may adversely affect plant growth. [Display omitted] • Both PVC and PLA MPs caused phytotoxicity and inhibited rice growth. • Both PVC and PLA MPs promoted enzyme activities in soil hotspot. • Enzyme activity showed high resistance to PVC in coldspots but low resistance to PLA in hotspots with different efficiencies. • MPs-induced changes in enzyme activities were associated with changes in bacterial co-occurrence structure. [ABSTRACT FROM AUTHOR]

The importance of root hairs and root radius for exudation and nutrient acquisition by plants is known mainly from nutrient solution studies. The in situ effects of root hairs and root radius on the spatial distribution of enzyme activity in the rhizosphere of various plants are unknown. Four plants with contrasting root morphology (maize, wheat, lentil and lupine) were chosen to test the effects of root hairs and root radius on the spatial distribution of β-glucosidase, cellobiohydrolase, leucine aminopeptidase and acid phosphatase. We combined zymography with enzyme kinetics to evaluate the effects of root hairs on the rhizosphere extent and on substrate turnover. The extent of enzyme activity in the rhizosphere of four plants ranged from 0.55 to 2 mm. The extent of β-glucosidase was 1.5 times broader (1.2 mm versus 0.8 mm) and the substrate turnover was 2-fold faster around wheat root regions with hairs than hairless locations. The rhizosphere extent relative to root radius and the enzyme activity per root surface area were plant and enzyme specific: the rhizosphere extent was 1.5–2 times broader and the enzyme activity was 2–8-fold higher in wheat (with thin roots and long root hairs) compared to maize, lentil and lupine. The rhizosphere extent of acid phosphatase (1.1–2.0 mm) was 1.5–2-fold broader than that of other enzymes (0.5–1.0 mm). For the first time, we showed that the rhizosphere extent relative to root radius was 20–100% broader and enzyme activity per surface area was 4–7-fold higher around thin roots (wheat) than around thick roots (maize). Moreover, the rhizosphere extent relative to root radius was 10–30% broader and enzyme activity per root area was 2–7 times higher around roots with long and dense hairs (lupine) than around roots with short and sparse hairs (lentil). We conclude that root hairs and root radius shape the rhizosphere: root hairs contributed mainly to the rhizosphere extent, while root radius more strongly affected the enzyme activity per root surface area. [ABSTRACT FROM AUTHOR]

The rhizosphere is the most dynamic hotspot of microbial activity in the soil. Despite these dynamics, the spatial pattern of many rhizosphere properties may remain stable because they are continuously reproduced in the changing environment. Low substrate concentration can strongly reduce the rate response of an enzymatic reaction subjected to increased temperature and is recognized as a canceling effect on enzyme temperature sensitivity. Carbon input from rhizodeposits affects C availability in the rhizosphere, and thus the enzyme activities responsible for organic matter decomposition, and their temperature sensitivities, upset the dynamics and stability of biochemical processes in the rhizosphere. However, it is unclear whether a canceling effect occurs in the rhizosphere. We studied temperature effects on chitinase and phosphatase during rice ( Oryza sativa L.) growth at 18 and 25 °C. The spatial distribution of enzyme activities was imaged using soil zymography and showed that the overall activities of these enzymes increased with temperature but decreased with rice growth. The temporal dynamics of hotspot areas were enzyme-specific. During growing days 14–30, hotspot areas decreased from 2-2.5% to 0.3–0.5% for chitinase, but increased from 2% to 6–7% for phosphatase. The distribution pattern of both enzymes shifted from being dispersed throughout the soil to being associated with the roots. For the first time, we showed the extent of rhizosphere enzyme activity in paddy soil and demonstrated that it is temporally stationary and independent of temperature. However, the temperature sensitivity of enzyme activities declined radically ( Q 10 ∼1.3–1.4) at the root surface compared to that of bulk soil ( Q 10 ∼1). We conclude that the spatio-temporal pattern of rhizosphere enzymatic hotspots is mainly affected by plant growth. High temperature sensitivity ( Q 10 > 1) at the root-soil interface for the tested enzymes revealed that warming will lead to faster nutrient mobilization in the rhizosphere than in root-free soil. [ABSTRACT FROM AUTHOR]

Land-use change, especially from forest to intensive agriculture, is negatively impacting soil quality and sustainability. Soil biological activities are sensitive indicators of such land-use impacts. We tested two hypotheses: i) land use and management practices affect microbial properties (microbial biomass and enzyme activities) in topsoil (0–20 cm), but have no effects in subsoil (20–100 cm); and ii) microbial properties in topsoil are highest in forest, followed by organic farming and then conventional farming. Total organic C and N contents as well as microbial biomass were significantly higher in the organic farming topsoil compared with conventional farming and forest. Except xylanase and acid phosphatase, enzyme activities (β-glucosidase, cellobiohydrolas, chitinase, sulfatase, leucine aminopeptidase and tyrosine aminopeptidase) were also higher in organic farming soil. Crop residues and rhizodeposits support higher microbial biomass, leading to enhanced enzyme activities in organic farming soil. Incorporation of rice stubble and limitation of available phosphorus explain the higher xylanase and acid phosphatase activities, respectively, in conventional farming soil. Litter removal leads to a deficiency of labile C and N, resulting in lower enzyme activities in forest soil. Total C and N contents were higher in subsoil under organic farming. Although there was no effect of land use on microbial biomass in subsoil, activities of most enzymes were higher under organic farming. Overall, our results indicate that land-use change significantly alters microbial properties in topsoil, with modest effects in subsoil. Microbial properties should be considered in environmental risk assessments and models as indicators of ecosystem disturbance caused by land-use and management practices. [ABSTRACT FROM AUTHOR]

Temperature effects on enzyme kinetics and on the spatial distribution of microbial hotspots are important because they are crucial to soil organic matter decomposition. We used soil zymography ( in situ method for the two dimensional quantification of enzyme activities) to study the spatial distributions of enzymes responsible for P (phosphatase), C (cellobiohydrolase) and N (leucine-aminopeptidase) cycles in the rhizosphere (living roots of maize) and root-detritusphere (7 and 14 days after cutting shoots). Soil zymography was coupled with enzyme kinetics to test temperature effects (10, 20, 30 and 40 °C) on the dynamics and localization of these three enzymes in the root-detritusphere. The percentage area of enzyme activity hotspots was 1.9–7.9 times larger and their extension was broader in the root-detritusphere compared to rhizosphere. From 10 to 30 °C, the hotspot areas enlarged by a factor of 2–24 and V max increased by 1.5–6.6 times; both, however, decreased at 40 °C. For the first time, we found a close positive correlation between V max and the areas of enzyme activity hotspots, indicating that maximum reaction rate is coupled with hotspot formation. The substrate turnover time at 30 °C were 1.7–6.7-fold faster than at 10 °C. The K m of cellobiohydrolase and phosphatase significantly increased at 30 and 40 °C, indicating low affinity between enzyme and substrate at warm temperatures. We conclude that soil warming (at least up to 30 °C) increases hotspot areas of enzyme activity and the maximum reaction rate (V max ) in the root-detritusphere. This, in turn, leads to faster substrate exhaustion and shortens the duration of hot moments. [ABSTRACT FROM AUTHOR]

Recycling of livestock manure in agroecosystems has been shown to enhance the sustainability of food production and reduce adverse environmental consequences from intensive crop-livestock systems. However, the effect of manure application on the associations between soil quality and ecosystem multifunctionality still remains poorly understood. Hereby, we used a five-year field experiment to investigate the effect of mineral and manure fertilization on soil quality, enzymatic stoichiometry, and ecosystem multifunctionality for both topsoil and subsoil (i.e. 0-20 cm and 20–40 cm). Manure alone and combined with mineral fertilization increased soil quality index by 49.5% and 70.1% in the topsoil, and by 67.5% and 26.6% in subsoil compared to no fertilization. Moreover, the manure application increased the C, N, and P acquisition enzyme activities, especially those for C and P cycling. Fertilization regimes affect enzymatic stoichiometry in the subsoil rather than topsoil. Manure application increased soil ecosystem multifunctionality in both top and subsoil by 2.1 and 0.4 times, respectively. Interestingly, the soil quality index was positively correlated with ecosystem multifunctionality regardless of fertilization regimes. Furthermore, random forest analysis showed that soil organic C and N content, available P, and microbial biomass were the main drivers of soil ecosystem multifunctionality. Conversely, mineral fertilization did not affect soil quality and enzyme activity in both soil layers, and thus did not change soil ecosystem multifunctionality. In conclusion, manure application fosters soil quality and has the potential to improve the soil multifunctionality, thereby providing an effective way to sustainable soil management and cleaner crop production. [Display omitted] • Manure addition increased soil quality and ecosystem multifunctionality. • Higher C and P enzyme activities promote ecosystem multifunctionality. • Fertilization regimes affect enzymatic stoichiometry in subsoil rather than topsoil. • Soil quality and ecosystem multifunctionality are co-driven by fertilization. [ABSTRACT FROM AUTHOR]

Glucose is one of the low molecular weight components of root exudates to mediate the cross-talk between plants and microbes, but less is known about their contribution to drought resistance of plants and root-associated microbiome. To fill this knowledge gap, we optimized the visualization of glucose exudation and coupled it with another in situ tool – soil zymography ‒ as well as destructive analysis of enzyme kinetics (β-glucosidase; acid phosphomonoesterase) and microbial biomass. This helped identify how microbial functionality ‒ affected by drought and P limitation ‒ will show more resistance in the hotspots of soybean rhizosphere (grown in the rhizoboxes for 10 weeks) associated with arbuscular mycorrhizal fungi (AMF) symbiosis than those without AMF. Drought reduced glucose exudation, mainly allocated to root tips, and narrowed the rhizosphere enzymatic hotspot by three times. However, AMF inoculation enhanced glucose exudation compared to non-mycorrhizal plants and enlarged enzymatic hotspot area by 53% under drought condition. Despite the 50% reduction in β-glucosidase and acid phosphomonoesterase activities owing to water deficit, AMF symbiont triggered up to 36% enzyme activities in correlation with the non-mycorrhizal ones. Therefore, the drought resistance of these two enzymes was enhanced by up to 63% in mycorrhizal plants. The biomass of microbial phosphorus increased by 45% under drought AMF-conditioned plants. We conclude that the cooperation between soybean and AMF induced the formation of favorable microsites around the root, specifically in overlapping localities between rhizosphere and mycorrhizosphere, characterized by enhanced glucose release, increasing rhizosphere expansion, high enzyme activities and shortened substrate turnover time. This, in turn, contributed to the stronger resistance of microbial functions (e.g., enzyme expression) to drought stress in the rhizosphere hotspots. Thus, in response to AMF inoculation and consequent high glucose availability, rhizosphere microorganisms increased P mining rate in those hotspots remaining active despite water scarcity. [Display omitted] • Glucose imaging has been optimized and adapted for the soil environment. • Glucose exudation is higher in the mycorrhizal plant roots than control under drought. • AMF inoculation enlarges enzymatic hotspot area under drought condition. • Drought resistance of enzymes is enhanced in mycorrhizal plants. [ABSTRACT FROM AUTHOR]

Understanding phosphorus (P) dynamics in the rhizosphere is crucial for sustainable crop production. P mobilization processes in the rhizosphere include the release of plant and microbially-derived protons and extracellular phosphatases. We investigated the effect of root hairs and soil texture on the spatial distribution and intensity of P mobilizing processes in the rhizosphere of Zea mays L. root-hair defective mutant (rth3) and wild-type (WT) grown in two substrates (loam, sand). We applied 2D-chemical imaging methods in custom-designed root windows installed in the field to visualize soil pH (optodes), acid phosphatase activity (zymography), and labile P and Mn fluxes (diffusive gradients in thin films, DGT). The average rhizosphere extent for phosphatase activity and pH was greater in sand than in loam, while the presence of root-hairs had no impact. Acidification was significantly stronger at young root tissue (<2 cm from root cap) than at older root segments (>4 cm from root cap) and stronger in WT than rth3. Accompanied with stronger acidification, higher P flux was observed mainly around young, actively growing root tissues for both genotypes. Our results indicate that acidification was linked to root growth and created a pH optimum for acid phosphatase activity, i.e., mineralization of organic P, especially at young root tissues which are major sites of P uptake. Both genotypes grew better in loam than in sand; however, the presence of root hairs generally resulted in higher shoot P concentrations and greater shoot biomass of WT compared to rth3. We conclude that soil substrate had a larger impact on the extent and intensity of P solubilization processes in the rhizosphere of maize than the presence of root hairs. For the first time, we combined 2D-imaging of soil pH, phosphatase activity, and nutrient gradients in the field and demonstrated a novel approach of stepwise data integration revealing the interplay of various P solubilizing processes in situ. [Display omitted] • In situ chemical imaging of pH, acid phosphatase activity, P and Mn flux in the rhizosphere of field grown maize. • Average rhizosphere extents were larger in sand than in loam. • Root hairs mostly did not increase rhizosphere extents of imaged parameters. • Young root tissue of wild-type maize was more acidic than of root hair-defective mutant rth3. • Acidification from young root tissue created pH optimum for acid phosphatase activity and P uptake. [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]

Climate change impacts soil microbial communities, activities and functionality. Nonetheless, responses of the microbiome in soil microenvironments with contrasting substrate availability in the rhizosphere to climatic stresses such as drought are largely unknown. To fill this knowledge gap, we coupled soil zymography with site-specific micro-sampling of the soil and subsequent high-throughput sequencing. This helped identify how the bacterial community structure and the genes encoding N-cycling enzymes (leucine aminopeptidase and chitinase) in rhizosphere hotspots and coldspots (microsites with activities in the range of bulk soil but localized within the rhizosphere) of maize respond to drought (20% WHC, two weeks). The elevated activities of leucine aminopeptidase and chitinase in rhizosphere hotspots were caused by the tight collaborative relationships between bacteria and their stable network structure rather than by any significant shift in bacterial community structure or enzyme-encoding gene copies. Despite the similarity in bacterial community structure in soil under drought and optimal moisture, functional predictions indicated the increased relative abundance of genera belonging to Actinobacteria capable of leucine aminopeptidase and chitinase production, especially Streptomyces , Nocardioides , Marmoricola, and Knoellia. Accordingly, the number of gene copies encoded by Actinobacteria for these two enzymes increased by 5.0–17% under drought. Among the bacteria with increased relative abundance under drought, Luedemannella played a crucial role in mediating nutrients and energy fluxes between bacteria. This was reflected in a 35–70% increase in leucine aminopeptidase and chitinase activities under drought. The resistance of enzyme activities to drought was higher in hotpots than that in coldspots. These results revealed that rhizosphere bacterial community composition remained stable, and that the number of gene copies encoded by Actinobacteria responsible for N-cycling enzymes increased under drought. The expected reduction of processes of N cycle was absent. Instead, bacteria increased N mining rate in those hotspots remaining active despite water scarcity. [Display omitted] • Soil zymography is combined with high-throughput sequencing • "Rhizosphere enzymatic coldspots" is for the first time localized • Enzyme activity in hotspots was more resistant than that in coldspots to drought • Bacterial community composition was stable between rhizosphere hot- and coldspots • Enzyme-related genes encoded by bacteria were affected by drought [ABSTRACT FROM AUTHOR]

Soil–plant interactions affecting nitrous oxide (N2O) are not well-understood, and experimental data are scarce. Therefore, a greenhouse experiment was conducted in a 3 × 3 full factorial design, comprising three mineral N fertilizer rates (0, 150 and 300 kg N ha−1) applied to monoculture swards and a binary mixture of Plantago lanceolata and Lolium perenne. The parameters measured included daily N2O emissions, aboveground (AG) and belowground biomass (BG), N and C yields, as well as leucine aminopeptidase (LAP) activity in the soil as an indicator for soil microbial activity. Nitrous oxide emission and LAP were measured using the static chamber method and fluorimetric microplate assays, respectively. Cumulative N2O emissions were about two times higher for P. lanceolata than L. perenne monoculture swards or the mixture (p < 0.05). The binary mixtures also showed the highest N use efficiency and LAP activity, which significantly (p < 0.05) correlated with the C concentration in the belowground biomass. Plantago lanceolata was generally ineffective at reducing N2O emissions, probably due to the young age of the swards. Among the biological factors, N2O emission was significantly associated with biomass productivity, belowground C yield, belowground N use efficiency and soil microbial activity. Thus, the results suggested belowground resource allocation dynamics as a possible means by which swards impacted N2O emission from the soils. However, a high N deposition might reduce the N2O mitigation potential of grasslands. [ABSTRACT FROM AUTHOR]