SSS4.2

Soil enzymes produced by microorganisms and plants are very sensitive to the variations in microclimate, e.g. aeration, and respond quickly to the induced changes. The majority of the enzyme assays are conducted under normal (temperature and air) conditions irrespectively of the origin of the environmental samples. However, it remains unclear how conditions of assays may affect results in anaerobic systems. In the present study, we have clarified this key gap in current methods by measuring the kinetics of phosphatase, β-glucosidase, and leucine aminopeptidase in paddy soil under aerobic and anaerobic conditions by means of a glovebox. Specifically, we quantified V_max and K_m in soil from three compartments in a rhizobox (top bulk (2-5 cm), rhizosphere, and bottom bulk (15-18 cm)) during rice growth. We demonstrate that the activities of three tested enzymes were significantly lower under aerobic conditions compared to anaerobic conditions at three consecutive dates of rice growth. Lower V_max values for phosphatase in top bulk soil and rhizosphere soil and β-glucosidase in top bulk soil, rhizosphere soil, and bottom bulk soil confirmed that aerobic conditions limited enzyme activities. For leucine aminopeptidase, although the difference in V_max values between anaerobic and aerobic conditions was not significant, the values always increased under anaerobic conditions compared to aerobic conditions. Compared with anaerobic conditions, the K_m values for phosphatase under aerobic conditions decreased by 10.11-22.78%. The maximum difference in the K_m values for β-glucosidase and leucine aminopeptidase between aerobic and anaerobic conditions was 30.93% and 40.53%, respectively. We conclude that enzyme activities of samples taken from the anaerobic or low-redox environment have to be assayed under anoxic conditions to avoid 10-40% underestimation (for V_max) due to suppression by oxygen.

How to cite: Wang, C., Dorodnikov, M., Blagodatskaya, E., and Dippold, M.: An improved method for extracellular enzyme assays in paddy soil: a comparative study under aerobic and anaerobic conditions, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-2180, https://doi.org/10.5194/egusphere-egu21-2180, 2021.

Alive plants and soil microorganisms are the influential sources of extracellular enzymes facilitating decomposition of polymeric organic compounds. Enzyme activities are especially intensive and spatially heterogeneous in the rhizosphere, where microorganisms are stimulated by rhizodeposition. Two-dimensional activity distribution of hydrolytic enzymes participating in transformation of soil organics in the distance gradients from the root can be visualized under UV light by zymography - by placing a fluorogenic substrate-saturated membrane on the soil surface. Functional traits of enzymes can be co-localized with spatial distribution of enzymatic activity by precise micro-sampling based on zymography. We used rhizobox experiment to visualize activity of β-glucosidase, leucine aminopeptidase, and phosphatase in the rhizosphere of wild type and root hairless mutant of Zea mays L. cultivated for 3 weeks. After precise micro-sampling, we determined kinetic parameters of enzymes: max potential activity and affinity to substrate in the rhizosphere gradients. Finally, we compared the correspondence of enzymatic activity determined by zymography and by kinetic approach. This work was conducted within the framework of the priority program 2089, funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project number: 403664478. Seeds of the maize were provided by Caroline Marcon and Frank Hochholdinger (University of Bonn).

Keywords: zymography, enzyme kinetic, maize, rhizosphere gradients

How to cite: Ghaderi, N., Guber, A., Khosrozadeh, S., Guliyev, V., and Blagodatskaya, E.: Functional traits of hydrolytic enzymes and visualized enzymatic activity in the rhizosphere gradients of Zea mays L., EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-5841, https://doi.org/10.5194/egusphere-egu21-5841, 2021.

This study was conducted within the framework of the DFG project SPP2089 “Rhizosphere Spatiotemporal Organization – a Key to Rhizosphere Functions” (project number 403641192).

As plant roots grow into the soil, the formation of biological gradients occurs at different spatial scales. It has been shown that plants recruit specific subsets of the soil bacterial community at their roots through excretion of mucilage at root tips and exudates at the sites of root hair formation. The promotion of or defense against certain bacterial taxa is also reflected in the composition of the protist communities that feed on bacteria.

Using high-throughput sequencing methods, we investigated emerging patterns in root gene expression in relation to bacterial and protozoan community structures. We found highly distinct root region specific patterns relating to differential root gene expression relating to growth, defense and transporter activity, as well as bacterial and protist (cercozoan) diversity. Root cap removal led to differently composed microbial communities, as well as a regulation of root genes relating to stress and defense. The lack of root hairs was only reflected in the amount of microbial carbon in soil and a small number of differentially expressed genes involved in cell wall processes.

We could show that the rhizosphere microbiome, is as dynamic as its environment. Root regions differentially affect microbial communities, which is also reflected in the expression of plant genes of categories relating to defense, immunity and stress. Our findings will further enhance our understanding of microbial root interactions at single root scale.

How to cite: Ganther, M., Rüger, L., Bonkowski, M., Heintz-Buschart, A., and Tarkka, M.: Integrated analysis of plant gene expression and bacterial and protozoan community composition reveals changes related to root zonation, root cap and root hair formation, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-7518, https://doi.org/10.5194/egusphere-egu21-7518, 2021.

The deposition of energy rich carbon sources released by plant roots during their growth fuels microbially driven ecosystem processes in soil, but there is a lack of understanding how microorganisms interact and collaborate. The objective of this research was therefore to characterize microbial networks as they assemble under the influence of plant roots. To identify the specific importance of root hairs, we compared the impact of a maize wild-type to a root-air defective mutant (rth3; (1).

The microbial community structure was analyzed by qPCR and 16S rRNA gene amplicon sequencing from soil DNA. In order to increase the probability of detecting truly interacting microbial partners as a basis for network analyses, we first evaluated a new protocol to obtain DNA from as little as 1 mg instead of the usual 250 mg soil samples, thereby approaching the aggregate level (2). While the diversity of bacterial 16S rRNA gene amplicons of 250-mg samples taken from the same soil was not distinct, DNA analyses from individual aggregates clearly differed from each other underlining that soil aggregates represent distinct microbial habitats.

Soil column experiments with maize grown in a loam soil (3) revealed distinct communities between rhizosphere and bulk soil. The community composition of individual aggregates showed more differences in bulk soil compared to rhizosphere. Less elaborated networks were seen in bulk soil and a profound effect of root hairs could be unravelled. Null model testing demonstrated that Actinobacteria were equally important for network connectivity independent of the root hair mutation, but for networks of the wildtype, Acidobacteria were essential for synergistic interactions and overall network structure. In contrast, Proteobacteria and Firmicutes connectivity became more important. The observed differences in community composition and interactions suggests carbon cycling, and perhaps other microbially-driven functions, are markedly affected by the presence of root hairs.

Utilizing maize root soil microcosms for studying soil zymography in the rhizosphere allowed to obtain soil samples from regions with distinct specific enzyme activities. In order to enhance the detection of actively metabolizing bacterial community members, we studied rRNA sequences and compared it to rRNA gene sequences from the same samples. Currently the data are under analysis.

References

(1) Wen, T-J, Schnable PS (1994) Analyses of mutants of three genes that influence root hair development in Zea mays (Gramineae) suggest that root hairs are dispensable. Am. J. Bot. 81, 833–842.

(2) Szoboszlay M, Tebbe CC (2020) Hidden heterogeneity and co-occurrence networks of soil prokaryotic communities revealed at the scale of individual soil aggregates. Microbiol. Open, e1144. DOI: 10.1002/mbo3.1144

(3) Vetterlein D et al. (2020) Experimental platforms for the investigation of spatiotemporal patterns in the rhizosphere – laboratory and field scale. J. Plant Nutr. Soil Sci., 000, 1–16 DOI: 10.1002/jpln.202000079

How to cite: Tebbe, C., Damini, D., Finn, D., Bilyera, N., Ganther, M., Szoboszlay, M., Tarkka, M., and Razavi, B. S.: Soil aggregate-based nucleic acid analyses of the impact of maize roots and their root hairs on the structural diversity of microbial communities, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-7620, https://doi.org/10.5194/egusphere-egu21-7620, 2021.

Plants interact with the rhizosphere microbiome via root exudates that consist of numerous metabolites serving as energy or carbon sources for microbial growth and as modulators of the uncountable rhizosphere interactions. The rhizosphere microbiome plays also an important role in plant health, growth, and productivity. Different drivers are known to shape the rhizosphere microbiome, but limited investigation exists whether there is a spatial variability in the microbiome along the root system (depth). The present study aimed to assess effects of potentially different drivers such as soil substrates, soil compartments (rhizosphere, and bulk soil), depths, and plant genotypes on bacterial/archaeal communities associated with two maize genotypes, root hair defective mutant (rth3), and the corresponding wild-type (WT). Experiments using maize genotypes rth3 and WT, grown on soil substrates loam, and sand under growth chamber, and field conditions were performed. Under growth camber conditions, the rhizosphere samples were harvested at twenty-two days after sowing the maize seeds from three different soil depths at 4.5 – 6.1 (GD1), 9.0 – 10.6 (GD2), and 13.5 – 15.1 (GD3) cm from soil surface. Under field conditions, analyses were carried out using both rhizosphere, and bulk soil samples taken at three developmental growth stages BBCH14, -19, and -59 of the maize plants; each from two depths at (0 – 20) FD1, and FD2 (20 - 40) cm from soil surface, except the BBCH14 (only samples from D1 were available). Bacterial/archaeal communities were analyzed by MiSeq Illumina sequencing of 16S rRNA gene fragments amplified from total community DNAs.

Under growth chamber conditions, we observed shifts in bacterial/archaeal diversity of maize rhizosphere at different depths as plant genotype- and soil substrate-dependent effects. Depth-dependent effects of maize rhizosphere (rth3/WT) on bacterial/archaeal compositions displayed high differences between GD1, and the GD3 on both soil substrates. The relative abundances of the bacterial phylum Proteobacteria were significantly higher at GD3 than GD1 for both plant genotypes on sand, but not on loam. Overall, the factor soil substrate was the strongest driver of bacterial/archaeal maize rhizosphere, followed by depth, and maize genotype.

Under field conditions, depths affected the rhizosphere bacterial/archaeal diversity only at the BBCH59 for WT grown on sand. Lower bacterial/archaeal diversity in soil substrates sand than loam was observed at both FD1 and FD2 in the rhizosphere, but not in bulk soil at all developmental growth stages of maize. The bacterial/archaeal diversity of both maize genotypes was not affected by developmental growth stages of maize on both soil substrates, and soil compartments. Depth gradients of bacterial/archaeal community composition in rhizosphere, and bulk soil displayed at BBCH59 on both soil substrates, and they were relatively higher on sand than loam (rhizosphere). Differences in relative abundances of the bacterial phyla Proteobacteria, and Actinobacteria between soil compartments, developmental growth stages of maize were observed mainly at FD1. Overall, factor soil compartment is the strongest driver of bacterial/archaeal communities followed by soil substrates, developmental growth stages and sampling depths for maize grown under field conditions.

How to cite: Yim, B., Ganther, M., Heintz-Buschart, A., Tarkka, M., Vetterlein, D., and Smalla, K.: Spatiotemporal organization of bacteria/archaea in maize rhizosphere, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-7890, https://doi.org/10.5194/egusphere-egu21-7890, 2021.

Root exudates are thought to play an important role in plant-microbial interactions. In return, soil bacteria can increase the bioavailability of soil minerals, which is typically decreasing in situations such as drought. Here we describe an exudate-driven microbial priming on Cupressus saplings grown outside in forest soil in custom-made rhizotron boxes. A 1-month imposed drought and inoculations with Bacillus subtilis and Pseudomonas stutzeri, bacteria species forest soil isolation, were applied in a factorial design. We revealed that both bacteria associated with Cupressus roots and were more abundant in rhizosphere than in bulk soil. Moreover, root exudation rate increased in inoculated trees under drought with >100 first identified metabolites from Cupressus roots. Among these metabolites, phenolic acid compounds, quinate, and others, were used as carbon and nitrogen sources by both bacterial species. Furthermore, soil phosphorous bioavailability was maintained only in inoculated trees, where a drought-induced decrease in leaf phosphorus and iron was prevented. We provide evidence that changes in exudation rate and composition under drought and bacteria inoculation, support the idea of root recruitment of beneficial bacteria. In turn, trees secreted further carbon source to the rhizosphere and hosted more bacteria, benefited from improved nutrition.

How to cite: Oppenheimer-Shaanan, Y., Jakoby, G., Starr, M. L., Karliner, R., Eilon, G., Itkin, M., Malitsky, S., and Klein, T.: A dynamic rhizosphere interplay between tree roots and soil bacteria under drought stress, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-8069, https://doi.org/10.5194/egusphere-egu21-8069, 2021.

Plants allocate an estimated 11% of the C that they fixate as root exudates, a complex mixture of compounds that helps engineer the plant’s subterranean habitat. Root exudates stabilize soil aggregates, improve water retention, and shape rhizosphere microbial community composition. Exudates are also thought to contribute to the formation of stable mineral-associated organic matter. However, the function and fate of exudates along the soil profile may differ. We hypothesize that in topsoils with highly active microbial populations and mineral surfaces saturated with organic matter, root exudates may be rapidly intercepted and assimilated by soil microbes, and later adsorbed to surfaces as microbial necromass. But in subsoils with low microbial activity, exudates may directly adsorb on unsaturated mineral surfaces. The magnitude of these divergent pathways can shape the role of root exudates in rhizosphere C cycling. However, little is known about how adsorption vs. decomposition processes at the root-soil interface control i) the chemical transformations of C occurring along the root-microbe-mineral pathway, and ii) the spatial distribution and heterogeneity of exuded and processed exudate C. Our objective was to investigate the effect of microbial activity, and reactive mineral surfaces on the spatial distribution and functional group chemistry of root exudates at the root-microbe-mineral interface.

We packed samples from O, A, B, and C horizons collected from a grassland Mollisol, into individual microcosms, and installed a porous microdialysis membrane which served as an artificial root. Through this root, we injected either root exudates collected from maize plants, or dissolved organic carbon extracted from plant litter collected at the site. This comparison allowed us to study the dynamics of organic inputs entering the soil profile from the litter layer vs. directly from the roots. We destructively sampled the microcosms and obtained intact cross sections containing the artificial root and surrounding mineral/pore structures at different time points throughout the experiment. Here, we will present results obtained using synchrotron-radiation FTIR-microscopy of the temporal evolution, and spatial distribution of organic matter functional group chemistry in an artificial rhizosphere.

How to cite: Shabtai, I., Lehmann, J., and Bauerle, T.: Investigating the chemical and spatial distribution of root carbon along the root-microbe-mineral pathway, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-8525, https://doi.org/10.5194/egusphere-egu21-8525, 2021.

Soil enzymes respond rapidly to changes in soil managements, and therefore are used as early and sensitive indicators of alteration in soil properties induced by tillage and additions of fertilizers. The aim of this work was to compare the effects of different tillage (no, minimum, and conventional tillage), fertilization and soil depth (0-30, 30-60 and 60- 90 cm) on the microbial biomass, enzyme activity and their relationship with soil nutrients in a semiarid Mediterranean agro-ecosystem. Growing and total microbial biomass decreased with depth together with the activities of β-glucosidase and N-acetyl-β-glucosaminidase presumably because of the reduced carbon and oxygen content in the deeper layers of soils. The fertilization stimulated fast-growing microorganisms with low affinity of enzyme systems to substrate, enhanced the growing microbial biomass and facilitated the turnover rate of soil organics. Under no tillage, all enzymes showed higher potential activity in top layers of fertilized plots as compared with non-fertilized ones. The minimum tillage practice increased the growing microbial biomass, and stimulated N- and P-acquiring enzymes due to  increased nutrients limitation. Parameters of microbial growth and enzyme kinetics are suitable indicators of microbial activity in semiarid Mediterranean agroecosystems.

How to cite: De Mastro, F., Traversa, A., Brunetti, G., and Blagodatskaya, E.: Enzymatic and microbial activities as influenced by tillage and fertilization in a semi-arid Mediterranean agroecosystem, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-8646, https://doi.org/10.5194/egusphere-egu21-8646, 2021.

Plant roots and soil microorganisms produce hydrolytic extracellular enzymes to acquire nutrients via transformation of organic matter. Microorganisms inhabit hydraulically active pores, being attached to their surfaces or to organic and mineral colloids in soil solution. Therefore, diffusion of enzymes due to Brownian motion is constrained by their interactions with the surfaces of soil particles dispersed in the solution. It is generally unknown to which extent the extracellular enzymes are associated with solid and liquid soil phases and whether enzyme motility is affected by the movement of colloids occurring in soil solution. Therefore, the goal of our study was to quantify enzyme transport in soils with contrasting properties. Transport of ß-glucosidase, acid-phosphatase, xylosidase and cellobiohydrolase was studied in undisturbed non-sterile columns of soils with three contrasting textures: sandy, sandy loam and loam. The colloids, microorganisms, and enzymes inherent for each soil were applied via soil suspensions to the tops of the undisturbed columns. The suspensions were prepared by dispersing 1 g of each soil in 100 ml of de-ionized water, followed by 30 min sedimentation. Approximately 2.5 pore volumes of the applied suspensions were passed through the columns with continuous collection of the effluent from the bottom of the columns. The effluent was analyzed for colloid contents and enzyme activities before and after removal of soil particles of size 1-10 μm by centrifugation. From 7 to 49% of applied colloids recovered from the columns with higher colloid retention capacity in finer textured soils. The enzyme activity and colloid content were the highest in the first portions of the effluent and decreased as more suspension passed the columns, suggesting presence of enzymes and colloids in soil pores readily available for convective transport. Removal of soil particles of size 1-10 μm from the effluents by centrifugation reduced enzyme activity by factors 2-5, which was much larger than reduction in the enzyme solutions free of colloids centrifuged at the same settings (24- 30%). Our results indicated that most enzymes are present and transported through soil pores convectively while attached to soil colloids. Support for this research was provided by the USDA NIFA Program (Award # 2019-67019-29361), by the NSF LTER Program (DEB 1027253) at the Kellogg Biological Station, by USDA NC1187 project, by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018409.

How to cite: Guber, A., Blagodatskaya, E., and Kravchenko, A.: Convective transport of enzymes through soil columns, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-9001, https://doi.org/10.5194/egusphere-egu21-9001, 2021.

Soil microorganisms preferably occupy intermediate-sized pores, which are the arena for most biochemical reactions due to high nutrient contents and beneficial air and water regimes in these pores. Extracellular enzymes produced by microorganisms for organic matter transformations are assumed to reside in the same pores. However, there is a lack of direct experimental evidence of enzymatic activity being associated with pores of particular sizes. In this study we measured activity of ß-glucosidase in soil pores Ø<14 μm, 14<Ø<164 μm and Ø<164 μm. Undisturbed soil cores (5 cm Ø, 5 cm height) were taken at continuous sorghum (G2), switchgrass (G5) and restored prairie (G10) treatments of KBS Great Lakes Bioenergy Research Center's Biofuel Cropping System Experiment. Soil cores were drained at 500 kPa, and undisturbed subsamples (0.8 cm Ø, 1 cm height) were taken from them and placed at ceramic plates connected to a vacuum system. The same quantities of 4-Methylumbelliferyl β-D-glucoside were applied to soil subsamples at vacuum corresponded to saturation of the three studied groups of pores and kept in soil for 30 min. Produced 4-methylumbelliferone (MUF) was then extracted from the soil. The results demonstrated that the enzyme activity increased in all groups of pores in the order G2<G5<G10, but was significantly different only between G2 and G10 treatments for Ø<14 μm pores (p<0.05). The enzyme activity was lower in Ø<14 μm pores than in the 14<Ø<164 and Ø<164 μm pores (p<0.05), with only numerically higher activity in Ø<164 μm compared to the 14<Ø<164 μm pores. Interconnectivity of pores drained at different matrix pressures likely results in an overestimation of the enzyme activity associated with Ø<14 μm pores and an underestimation of that associated with 14<Ø<164 μm pores, hence the observed differences among the pore sizes are likely smaller than the actual pore effects. Thus, the results support our earlier observations and confirm that the activity of extracellular enzymes is higher within intermediate-sized pores than within fine pores. Support for this research was provided by the USDA NIFA Program (Award # 2019-67019-29361), by the NSF LTER Program (DEB 1027253) at the Kellogg Biological Station, by USDA NC1187 project, by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018409.

How to cite: Kravchenko, A. and Guber, A.: Whether enzyme activity is the same in different soil pores, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-9009, https://doi.org/10.5194/egusphere-egu21-9009, 2021.

Due to global warming, future agriculture will have to face increasing temperatures, more frequent and extreme drought events and consequently water and nutrient scarcity. Thus, it is necessary to improve our understanding of how plants deal with dry conditions. Since there is still a lack of knowledge concerning below ground feedbacks of plants to drought, we are particularly interested in the response of below ground organs to soil drying.

Hence, the objective of our study was to determine morphological responses of roots and root hairs to soil drying in situ.
For this purpose, we have grown maize plants (Zea maize wildtype) in seedling holder microcosms for 8 days before harvesting and performing high-resolution synchrotron X-ray CT in order to visualize root compartements as well as the elongated root hairs (Koebernick et al. 2017). The segmented images served as basis for the quantification of our observations.

The results revealed that not only roots (Carminati et al. 2012) but also root hairs lose turgidity under dry soil conditions. This shrinkage of hairs occurs at high soil water potentials and reduces the surface and soil contact area of roots tremendously. Root hair shrinkage is the first step in a sequence of responses to progressive soil drying. The follow up processes within this sequence are the formation of cortical lacunae and root shrinkage resulting in air filled gaps at the root-soil interface. Severe cavitation within the xylem was not observed at the corresponding soil water potentials meaning that xylem embolism occurs at even lower potentials. This leads to the conclusion that there is a severe loss of root-soil contact and consequently of hydraulic conductivity at the root-soil interface before xylem cavitates and reduces water as well as nutrient fluxes in the radial root direction.
As not only roots but also root hairs take up nutrients and release exudates (Holz et al. 2017), they are assumed to be an important trait of the rhizosphere for both nutrient uptake and microbial activity. Furthermore, they increase the radial extent of the rhizosphere and although it is not yet clear if shrunk root hairs are inactive in exudation and nutrient uptake, their enormous shrinkage due to soil drying might limit rhizosphere processes.

In summary, losses of root-soil contact due to root and particularly root hair shrinkage are profound and occur at high water potentials.

References

• Carminati, A., Vetterlein, D., Koebernick, N., Blaser, S., Weller, U., & Vogel, H.-J. (2012). Do roots mind the gap? Plant and Soil, 367(1–2), 651–661. https://doi.org/10.1007/s11104-012-1496-9
• Holz, M., Zarebanadkouki, M., Kuzyakov, Y., Pausch, J., & Carminati, A. (2017). Root hairs increase rhizosphere extension and carbon input to soil. Annals of Botany, 121(1), 61–69. https://doi.org/10.1093/aob/mcx127
• Koebernick, N., Daly, K. R., Keyes, S. D., George, T. S., Brown, L. K., Raffan, A., Cooper, L. J., Naveed, M., Bengough, A. G., Sinclair, I., Hallett, P. D., & Roose, T. (2017). High-resolution synchrotron imaging shows that root hairs influence rhizosphere soil structure formation. New Phytologist, 216(1), 124–135. https://doi.org/10.1111/nph.14705

How to cite: Duddek, P., Ahmed, M., Koebernick, N., Ohmann, L., Lovric, G., and Carminati, A.: Loss of root-soil contact due to root and root hair shrinkage, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-15913, https://doi.org/10.5194/egusphere-egu21-15913, 2021.