Search

Recent policies and silvicultural management call for forest regeneration that involve the selection of tree species able to cope with low soil nutrient availability in forest ecosystems. Understanding the impact of different tree species on the rhizosphere processes (e.g., enzyme activities) involved in nutrient mobilisation is critical in selecting suitable species to adapt forests to environmental change. Here, we visualised and investigated the rhizosphere distribution of enzyme activities (cellobiohydrolase, leucine-aminopeptidase, and acid phosphomonoesterase) using zymography. We related the distribution of enzyme activities to the seedling root morphological traits of European beech (Fagus sylvatica) and Norway spruce (Picea abies), the two most cultivated temperate tree species that employ contrasting strategies in soil nutrient acquisition. We found that spruce showed a higher morphological heterogeneity along the roots than beech, resulting in a more robust relationship between rhizoplane-associated enzyme activities and the longitudinal distance from the root apex. The rhizoplane enzyme activities decreased in spruce and increased in beech with the distance from the root apex over a power-law equation. Spruce revealed broader rhizosphere extents of all three enzymes, but only acid phosphomonoesterase activity was higher compared with beech. This latter result was determined by a larger root system found in beech compared with spruce that enhanced cellobiohydrolase and leucine-aminopeptidase activities. The root hair zone and hair lengths were significant variables determining the distribution of enzyme activities in the rhizosphere. Our findings indicate that spruce has a more substantial influence on rhizosphere enzyme production and diffusion than beech, enabling spruce to better mobilise nutrients from organic sources in heterogeneous forest soils. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

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]

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]

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]

Anecic earthworms such as Lumbricus terrestris create effective channels for the translocation of organic substances, nutrients (N, P) and water between top- and subsoil. However, studies on localized and linked microbial activities with the biochemical mechanisms within drilosphere are missing. To clarify the enzymatic mechanisms, zymography was combined with 14C imaging by placing 14C-labeled wheat litter on the surface of mesocosms with top- and subsoil to feed earthworms. Cellobiohydrolase and acid phosphatase activities were compared in drilosphere and bulk soil to test two hypotheses: i) a positive spatial correlation between 14C images and enzyme activities at burrow edges reflects the interactions between substrate distribution and microbial activities; ii) the drilosphere has higher enzyme activities and faster substrate turnover compared to bulk soil independent on depth. In line with the first hypotheses, the localization of substrates (14C) overlapped with enzyme activities for 68%. Spatial distribution of enzyme activities had stronger gradients within 2 mm of burrow edges in topsoil and 4 mm in subsoil. The mineralization of the wheat litter was faster in drilosphere than in the bulk soil. In contrast to our second hypothesis, the substrate turnover was depth dependent: 14CO 2 efflux was fourfold faster in topsoil than subsoil drilosphere. The decline of enzyme activities with depth, accompanied by decreasing catalytic efficiency, implies microbial production of more efficient enzymes in top-than in the subsoil. We conclude that interactions between enzymes and substrates are more tight in the drilosphere, making it microbial hotspots. Thus, earthworm activities lead to microbial localization and acceleration of litter decomposition and C turnover in drilosphere. Therefore, both direct hotspot and indirect (soil heterogeneous content) effects need to be explicitly considered when attempting to model and predict how bioturbation evens will alter ecosystem dynamics and ecological development of (micro)habitats. • Coupling 14C imaging and zymography to illuminate enzyme distribution and C translocation in earthworm burrows. • Extent of enzyme distribution was narrower in topsoil than subsoil. • Hotspots of microbial activities 68% overlapped with 14C hotspots. • Favored microbial activities in earthworm burrows induced faster turnover of plant litter compared to bulk soil. [ABSTRACT FROM AUTHOR]