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

It is well documented that microbial biomass increases during winter in cold mountain or tundra ecosystems, but the cause and mechanism of such accumulation is unclear. Results from a grassland in Iceland demonstrated that the microbial biomass carbon (MBC, measured by the fumigation-extraction method) increased in winter, while microbial DNA content remained constant. We thus hypothesized that this accumulation of microbial biomass during the cold season is driven by the decrease in temperature, that increases the carbon storage of individual cells, but not by an increase in microbial cell numbers.To test this hypothesis, we conducted a laboratory incubation experiment with soils from a grassland in Iceland sampled before the onset of winter in early October (around 9 °C). We then exposed the soils to decreasing temperatures (0.5 °C, 3 °C, 6 °C and 9 °C) over five months. We analyzed microbial biomass carbon (MBC) and quantified the DNA content. Over the course of five months, we found higher MBC values at cool temperatures compared to warm conditions. As expected, cooling did not affect the DNA content, leading to a significantly higher MBC to DNA ratio when soils were incubated at 0.5 °C compared to 9 °C. This indicates that numbers of microbial cells did not change across temperatures, but that microbes at lower temperatures stored more carbon. We also found similar patterns in soils collected at different time points in the field. Furthermore, we estimated microbial DNA production, i.e., growth rates, by measuring the incorporation of 18O from labelled water into DNA. We observed lower microbial growth rates under field conditions in winter, indicating that increasing biomass carbon was not due to increased growth and that growth and turnover was balanced at all temperatures. Instead, we suggest that carbon uptake (which was decreased at lower temperatures) was less affected by cold temperatures than growth, so that microbial carbon could accumulate. We also verified this pattern in growth and carbon uptake rates with decreasing temperatures in the laboratory incubation experiment.Decreasing growth (cell division) and turnover rates with decreasing temperatures, at a lower but sustained carbon uptake rate, suggest that the cell size of soil microorganisms may increase when exposed to cooling. We will show and discuss first results from measurements with a suspended microchannel resonator (SMR), that, together with microscopic imaging allows to assess the mass (size) of individual cells of microorganisms at different temperatures.

The Arctic is warming at a faster rate than the rest of the planet due to climate change. The warmer temperatures are causing, among other effects, the permafrost to thaw. When ice-rich permafrost thaws, thermokarst features form due to subsidence of the ground surface and the creation of dynamic depressions, basins, and lakes. As a result, the hydrological cycle in these latitudes is intensifying, causing an increase of nutrients and organic carbon in surface waters. Such impacts on freshwaters affect microbial community composition, and thus, these systems are good sentinels to study processes in primary ecological succession related to ecosystem processes such as productivity and greenhouse gas (GHG) emissions. This study aims to improve the current understanding of microbial processes leading to release of GHG in thermokarst lakes. To that end, we sampled a total of 12 thermokarst basins in August 2022 along a latitudinal gradient (67ºN - 69ºN) in the Northwestern Territories (Canada). The basins were selected so that half were in the taiga biome and half in the tundra biome. In addition, based on satellite images and in-field observations, half of the lakes sampled were in expansion and the other half were undergoing drainage. Water samples were collected for the analysis of GHGs (CH4, CO2, N2O), major ions, dissolved nutrients (organic C, δ13C-DOC, organic and inorganic -N) and microbial community composition (16S rRNA gene metabarcoding). We used Ar-corrected gas saturation of each GHG as a proxy of net metabolic changes. Our first results show that both expanding and shrinking lakes were strongly oversaturated in CH4 andCO2, slightly saturated in N2O, and slightly undersaturated in O2, pointing out higher respiration activity than primary production. Microbial mineralization of organic matter was used as a proxy for GHG production. We found higher concentrations of dissolved organic C in shrinking lakes compared to expanding ones. Following the latitudinal gradient (i.e. biomes), higher temperatures were found in lakes sampled in the taiga compared to those located in tundra, together with deeper permafrost table depths. In surface waters, pH values and dissolved O2 concentrations were significantly higher in tundra lakes compared to taiga lakes, probably as a result of lateral DOM fluxes in more productive ecosystems (i.e. boreal forests). Differences between microbial communities in both biomes are therefore expected, which we will verify once the ongoing sequencing analyses are available. Our study advances the current knowledge of GHG dynamics in thermokarst lakes and helps to predict future effects of climate change impacts in northern latitudes.

The Arctic warms four times faster than the global average, resulting in widespread permafrost thaw. Organic matter that was stored in permanently frozen soil for up to millennia now becomes available to microbial decomposition. Warming might also alter microbial community composition and physiology and thus change the decomposition potential of soils. Our current knowledge about permafrost soil organic matter (SOM) composition and decomposition is limited, particularly in regard to the heterogeneity of permafrost landscapes, thus hampering our ability to predict possible permafrost soil feedbacks to climate change. The objective of this study was to characterize SOM and microbial community composition of the active layer and the upper permanently frozen soil from permafrost-affected polygonal lowland tundra.We collected more than 80 soil samples from four different soil layers (organic, mineral, cryoturbated, permanently frozen) from three developmental stages of ice-wedge polygons (low-center, flat-center, high-center polygons) in NW Canada, and analyzed organic matter composition by a pyrolysis-GC/MS fingerprinting approach and microbial community composition by amplicon sequencing of the 16S rRNA gene (bacteria, archaea) and the ITS1 region (fungi).Our results suggest that the spatial heterogeneity of permafrost soils is not only reflected in soil physical parameters, but also in the chemical composition of organic matter and the composition of microbial communities. The organic soil layer comprised both the highest microbial diversity and the most diverse SOM composition. The distribution of major compound classes (carbohydrates, lignins, lipids, N-compounds, phenols & aromatics) differed between organic, mineral, cryoturbated and permanently frozen organic matter. This pattern followed a gradient from low to high organic matter degradation with soil depth. Soil organic matter composition also differed among polygon types, indicating different decomposition pathways, likely depending on differences in vegetation and soil water availability. We also found distinct microbial communities for soils from low-center polygons, possibly driven by prevailing anoxic conditions in this landscape unit. Bacterial and archaeal communities differed among all soil layers, while only fungal communities from the organic soils differed from the other layers.The observed differences in SOM and microbial community composition among soil layers and polygon types highlight the importance of considering spatial heterogeneity when studying permafrost soils. Moreover, our results might help to explain observed differences in microbial decomposition patterns on different spatial scales and emphasize the need to include aspects of permafrost soil heterogeneity to finetune current ecosystem and climate models.This study is part of the EU H2020 project “Nunataryuk”.

Heterogeneity (spatial, temporal chemical and biological) is a fundamental property of soils. Although it is rarely explicitly accounted for in models of soil microbial functioning, it is a determinant of microbial access to substrate and therefore of microbial activity. Microbial adaptation to heterogeneity is also likely to play a significant role in determining microbial activity and therefore C persistence in soil. A more developed understanding of heterogeneity and how microbial communities interact with their heterogenous environment can help us better understand the mechanisms that regulate microbial activity and soil C dynamics, as well as offer potential avenues for upscaling. In this presentation I will show how microbial communities have adapted to spatial and molecular heterogeneity at the microbial scale and, through the use of a spatial explicit model, how spatial and molecular heterogeneity interact to reduce decomposition. Pore scale heterogeneity affects the distribution of both decomposers and organic matter. Using a stable isotope approach, I will show that, although there does not appear to be a clear relationship between microbial decomposer composition and pore size, a simple relationship emerges between pore size and microbial decomposition of organic substrate. As the pore size distribution of soils can be deduced from pedo-transfer functions, this relationship may provide a more mechanistic basis for the representation of moisture effects on C dynamics in larger scale models.

The last decades were characterized by rising temperatures, enhanced atmospheric CO2 concentrations, and by an increasing frequency of extreme events such as drought. Soil microorganisms are major drivers of biogeochemical processes, yet the effects of climate change in shaping microbial communities remain poorly understood.To address this knowledge gap, we examined how future climate conditions (combined +300 ppm CO2 and +3 °C warming, relative to ambient) and drought, alone and in combination, affect microbial community composition throughout the vegetation period in a sub-montane managed grassland (‘ClimGrass’ experiment; Styria, Austria). We combined amplicon sequencing of bacteria, archaea, and fungi with droplet digital PCR to perform quantitative microbiome profiling of seasonal and drought-legacy effects on soil microbial communities.Drought strongly shaped the bacterial/archaeal and the fungal community structure during peak drought conditions, and this effect could still be detected two and fourteen months after ending drought by rewetting and removing rain-out shelters. In comparison, future climate conditions were observed to exert less pressure on the structure of bacterial/archaeal and fungal communities. Interestingly, abundances of members of Actinobacteria and Bacteroidota for bacteria, as well as Cladosporiaceae and Phaeosphaeriacea for fungi (amongst others) significantly increased during peak drought. Our findings suggest that drought can have immediate and lasting effects on the soil microbial community structure by contributing to the establishment of drought-tolerant microbial communities.

Arbuscular mycorrhizal fungi (AMF) form mutualistic associations with roughly 70% of vascular plant species, supporting the nutrient acquisition of their host plants and deriving carbon in return. AMF and plant communities are linked to each other by host-specificity and the ecological selection of favorable nutrient and carbon trading strategies. Changing soil nutrient availabilities can affect both plant and AMF communities directly and also indirectly via the response of their partners. We aimed to elucidate the combined response of AMF (belowground) and plant (aboveground) community compositions to changing soil nutrient availabilities.We sampled soil and roots from a long-term nutrient deficiency experimental grassland in Admont (Styria, Austria). The grassland plots have been fertilized with different combinations of nitrogen (N), phosphorus (P), and potassium (K) over 70 years. Aboveground biomass cuts were removed three times each year, leading to long-term deficiencies of nutrients not replaced by fertilizers. Soil and root AMF community compositions were measured by DNA and RNA amplicon sequencing of the 18S rRNA gene. In addition, we assessed the plant community composition of the sampled roots by amplicon sequencing of the chloroplast rbcL (RuBisCo large subunit) gene region, and visually recorded the plant community composition on each investigated plot.Our results demonstrate that N and P deficiencies influenced soil AMF community composition, whereas K deficiency had a major impact on root AMF community composition. Interestingly, the plant community composition was affected by N and P, similar to the soil AMF community composition. Both, soil and root AMF community compositions were significantly correlated to plant community composition across all treatments, the correlation was however stronger for soil AMF communities (R2 = 0.55, p< 0.001). By using bipartite network analysis, we identified several fungus-plant pairs that responded consistently to treatments.Our results indicate that the response of grasslands to nutrient deficiencies is potentially driven by strong feedbacks between plant and belowground AMF community compositions. We here demonstrate that the known interactions between grassland plants and AMF - which are often investigated from a single plant or monoculture perspective - are major drivers of how diverse plant community compositions will respond to environmental change, such as fertilization. In conclusion, considering the ecology of the subsurface AMF communities may strongly benefit our understanding of plant communities in a future environment.

An increase in fire-prone conditions in non-fire adapted regions is rooted in climatic and anthropogenic changes. Such pyrogeographical shifts are observable, for example, in alpine regions. In 2021, Austria, experienced a fire larger than 100 ha for the first time in a century in the Schneeberg-Rax mountain region. In depth understanding of post-fire effects on carbon cycling at such non-fire adapted sites is still scarce. To help close this knowledge gap, post-fire changes were investigated at the abovementioned site, including soil organic matter composition and soil chemical conditions. Samples were taken immediately after the fire, 3 months, 6 months and 12 months thereafter from four sampling sites. Selected sites consisted of 1. a pine forest affected by a crown fire, 2. a pine and beech mixed forest affected by a surface fire, and two non-fire affected controls with similar site conditions (vegetation, slope, altitude, and exposition). Samples were analyzed for pH, carbon content, elemental composition, leachable dissolved organic carbon and trace elements, organic matter composition, and environmentally persistent free radical concentrations. pH increased after the fire at both sites investigated. This increase was the strongest (up to 1.5 units) immediately after the fire but was still substantial 1 year after the fire. Carbon contents decreased approximately 2fold in the crown fire affected soil compared to the control soil, but remained similar between surface fire affected soil and the respective control. However, aromaticity of bulk carbon and the leachable fraction increased in both fire-affected soils, which can be related to the formation of pyrogenic carbon during the fire. Pyrogenic carbon is a highly aromatic and recalcitrant carbon pool produced during incomplete combustion of biomass. Pyrogenic carbon can also contain substantial amounts of environmentally persistent free radicals (EPFR), which can form reactive oxygen species, which can induce oxidative stress on microbiota. Our EPFR measurements showed an increase by at least 1.5 orders of magnitude of EPFR in fire affected soils. This study suggests that changes in soil carbon cycling can be expected following fires in non-adapted alpine forests.

Soil harbours a huge diversity of soil microbes. One reason for this is thought to be its small-scale chemical and physical heterogeneity, which offers innumerable different parallel habitats and thus niches for the development of microbial communities. However, while microbial diversity and soil microbial composition are usually assessed using gram-sized homogenized soil samples, knowledge of how microbes are actually spatially organized at smaller scales is lacking. This not only hampers our ability to link microbial communities to their chemical environment or to functions they may mediate, but also limits our understanding of potential scale-dependent drivers of soil organic matter turnover. Here, we investigated fungal and bacterial community composition together with selected biogeochemical parameters across different spatial scales in a Beech forest soil. We picked around 200 individual two mm-sized soil aggregates, i.e. small soil clumps that sticked together and could be collected with a tweezer, from fourty 35 ml-sized soil cores (i.e. 4-5 aggregates per core) in two different soil depths across a 800 m2 forest area. The remaining soil cores were homogenized. We carried out amplicon sequencing of the 16S rRNA gene (for bacteria/archea) and the ITS region (for fungi) from individual soil aggregates and the homogenized soil cores, and additionally measured C, N, δ15N and δ13C from both sample types. Microbial community compositions of individual aggregates were highly distinct from each other, and from their composite ‘parent’ soil core, exemplifying a high spatial hetereogeneity of microbial communities at that small scale. Delta13C values were constantly higher in individual aggregates compared to their composite ‘parent’ core, indicating that they were relatively enriched in C that already underwent microbial recycling. We found a striking correlation between C concentration and δ13C values across all individual aggregates, suggesting that the aggregates remain intact long enough to allow for continued degradation and recycling, and hence 13C enrichment of the encapsulated C compounds. The variations of C concentration and δ13C across individual aggregates are partially explained by variations in community composition at this small scale. Overall our data shows that mm-sized soil aggregates host distinct microbial communities which may be linked to a certain recycling state of C. This suggests that aggregates are pieces of soil that stick together for a significant amount of time, allowing them to act as functional units of soil C degradation, and possibly as ‘evolutionary incubators’, as was previously hypothesized. We conclude that investigating microbial community structure and distribution at these small scales in the soil offer a promising way forward to better understand possible links between microbial community composition and soil functions.

Cyclic GMP (cGMP) is a second messenger that regulates numerous physiological and pathophysiological processes. In recent years, more and more studies have uncovered multiple roles of cGMP signalling pathways in the somatosensory system. Accumulating evidence suggests that cGMP regulates different cellular processes from embryonic development through to adulthood. During embryonic development, a cGMP-dependent signalling cascade in the trunk sensory system is essential for axon bifurcation, a specific form of branching of somatosensory axons. In adulthood, various cGMP signalling pathways in distinct cell populations of sensory neurons and dorsal horn neurons in the spinal cord play an important role in the processing of pain and itch. Some of the involved enzymes might serve as a target for future therapies. In this review, we summarise the knowledge regarding cGMP-dependent signalling pathways in dorsal root ganglia and the spinal cord during embryonic development and adulthood, and the potential of targeting these pathways. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Enzymes are secreted by microbial cells into the soil to catalyze the acquisition of carbon or nutrients like nitrogen and phosphorus from soil organic matter. Apart from microorganisms and soil fauna, roots also secrete enzymes to mobilize nutrients from the soil nutrient pool. Thus, living plants and microorganisms are considered the main sources of soil enzymes in agroecosystems. To understand how exo-enzymes are distributed in soil microhabitats, and whether enzymatic activity is higher in soil solution or on particle surfaces, we visualized enzymatic activity at the corresponding scale. Visualization of enzymatic activity links microbial functioning to localization in heterogeneous soil habitats. To assess enzymatic reactions in soil at the microscopic level, we developed a micro-zymography approach based on fluorogenically-labeled substrate (phosphomonoesterase) in the rhizosphere soil of Zea mays L. For this, first we compared different fixatives required to prevent sample drying and found super transparent silicon as the most appropriate one. Then we evaluated micro-zymography i) on individual soil aggregates, ii) on thin layers of aggregates (≈ 500 µm) to assess the dynamics of phosphomonoesterase activity, and iii) on maize roots under laser scanning microscope. The results demonstrated that the main fluorescence signal shifted from the soil solution to the interface between the soil solution and aggregates within 30 min after substrate addition and was finally only detectable on the surface of a few aggregates. This was probably due to higher microbial abundance and enzymatic activity on the soil aggregates compared to the soil solution. The enzymatic activity appeared patchy on the aggregate surfaces indicating heterogeneous distribution of microorganisms. Similarly, a patchy distribution of enzymatic activity was detected on maize root surfaces. This work was conducted within the framework of the priority program 2089 “Rhizosphere spatiotemporal organization – a key to rhizosphere functions”, 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: exo-enzyme, micro-zymography, fixative

Climate change threatens the Earth’s biggest terrestrial organic carbon reservoir: permafrost soils. With climate warming, frozen soil organic matter may thaw and become available for microbial decomposition and subsequent greenhouse gas emissions. Permafrost soils are extremely heterogenous within the soil profile and between landforms. This heterogeneity in environmental conditions, carbon content and soil organic matter composition, potentially leads to different microbial communities with different responses to warming. The aim of the present study is to (1) elucidate these differences in microbial community compositions and (2) investigate how these communities react to warming.We performed short-term warming experiments with permafrost soil organic matter from northwestern Canada. We compared two sites characterized by different glacial histories (Laurentide Ice Sheet cover during LGM and without glaciation), three landscape types (low-center, flat-center, high-center polygons) and four different soil horizons (organic topsoil layer, mineral topsoil layer, cryoturbated soil layer, and the upper permanently frozen soil layer). We incubated aliquots of all soil samples at 4 °C and at 14 °C for 8 weeks and analyzed microbial community compositions (amplicon sequencing of 16S rRNA gene and ITS1 region) before and after the incubation, comparing them to microbial growth, microbial respiration, microbial biomass and soil organic matter composition.We found distinct bacterial, archaeal and fungal communities for soils of different glaciation history, polygon types and for different soil layers. Communities of low-center polygons differ from high-center and flat-center polygons in bacterial, archaeal and fungal community compositions, while communities of organic soil layers are significantly different from all other horizons. Interestingly, permanently frozen soil layers differ from all other horizons in bacterial and archaeal, but not fungal community composition.The 8-week incubations led to minor shifts in bacterial and archaeal community composition between initial soils and those subjected to 14 °C warming. We also found a strong warming effect on the community compositions in some of the extreme habitats: microbial community compositions of (i) the upper permanently frozen layer and of (ii) low-center polygons differ significantly for incubations at 4 °C and 14 °C. Yet, the lack of a community change in horizons of the active layer suggests that microbes are adapted to fluctuating temperatures due to seasonal thaw events.Our results suggest that warming responses of permafrost soil organic matter, if not frozen or water-saturated, may be predictable by current models. Process changes induced by short-term warming can be rather attributed to changes in microbial physiology than community composition.This work is part of the EU H2020 project “Nunataryuk”.

Linking soil functions to microbial community structure is arguably one of the greatest challenges in soil ecology, presumably due to the structural complexity and heterogeneity of soil across scales and time, as well as the large number of microbial taxa present. To overcome these impediments, we here introduce a model soil ecosystem - the “Synthetic Soil” - which allows soil structure and microbial community composition to be varied separately, to disentangle the complex relationship. The Synthetic Soil consists of a mixture of sterilized primary and secondary soil minerals and organic matter of plant and microbial origin, which together constitute the artificial soil matrix. This abiotic soil matrix is then inoculated with an in-silico designed, minimal microbial community, composed of 12 selected soil bacteria and fungi.We demonstrate the applicability of the approach, by incubating the Synthetic Soil in a sterile environment for five weeks. During this period, an actively growing soil community established, indicated by stable respiration rates, increasing DNA- and ammonium concentrations, depletion of dissolved organic carbon, and by changes in relative abundances of the community members. Additionally, the minimal community was actively decomposing soil organic matter by the production of extracellular enzymes. In conclusion, the Synthetic Soil approach developed in this study, allows the construction of powerful and modifiable model ecosystems, which will make it possible to link soil functions to microbial community structure and thus to address fundamental questions of soil ecology.

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

Climate change is altering the frequency and severity of drought events. Recent evidence indicates that drought may produce legacy effects on soil microbial communities. However, it is unclear whether precedent drought events lead to ecological memory formation, i.e., the capacity of past events to influence current ecosystem response trajectories. Here, we utilize a long-term field experiment in a mountain grassland in central Austria with an experimental layout comparing 10 years of recurrent drought events to a single drought event and ambient conditions. We show that recurrent droughts increase the dissimilarity of microbial communities compared to control and single drought events, and enhance soil multifunctionality during drought (calculated via measurements of potential enzymatic activities, soil nutrients, microbial biomass stoichiometry and belowground net primary productivity). Our results indicate that soil microbial community composition changes in concert with its functioning, with consequences for soil processes. The formation of ecological memory in soil under recurrent drought may enhance the resilience of ecosystem functioning against future drought events., Legacies of past ecological disturbances are expected but challenging to demonstrate. Here the authors report a 10-year field experiment in a mountain grassland that shows ecological memory of soil microbial community and functioning in response to recurrent drought.

This study evaluates the performance of the antigen-based anterior nasal screening programme implemented in all Austrian schools to detect SARS-CoV-2 infections. We combined nationwide antigen-based screening data obtained in March 2021 from 5,370 schools (Grade 1–8) with an RT-qPCR-based prospective cohort study comprising a representative sample of 244 schools. Considering a range of assumptions, only a subset of infected individuals are detected with the programme (low to moderate sensitivity) and non-infected individuals mainly tested negative (very high specificity).

BackgroundThe role of schools in the SARS-CoV-2 pandemic is much debated. We aimed to quantify reliably the prevalence of SARS-CoV-2 infections at schools detected with reverse-transcription quantitative polymerase-chain-reaction (RT-qPCR).MethodsThis nationwide prospective cohort study monitors a representative sample of pupils (grade 1-8) and teachers at Austrian schools throughout the school year 2020/2021. We repeatedly test participants for SARS-CoV-2 infection using a gargling solution and RT-qPCR. We herein report on the first two rounds of examinations. We used mixed-effect logistic regression to estimate odds ratios and robust 95% confidence intervals (95% CI).FindingsWe analysed data on 10734 participants from 245 schools (9465 pupils, 1269 teachers). Prevalence of SARS-CoV-2 infection increased from 0.39% at round 1 (95% CI 0.28-0·55%, 29 September-22 October 2020) to 1·39% at round 2 (95% CI 1·04-1·85%, 10-16 November). Odds ratios for SARS-CoV-2 infection were 2·26 (95% CI 1·25-4·12, P=0·007) in regions with >500 vs. ≤500 inhabitants/km2, 1·67 (95% CI 1·42-1·97, PInterpretationThis monitoring study in Austrian schools revealed SARS-CoV-2 infection in 0·39%-1·39% of participants and identified associations of regional community incidence and social deprivation with higher prevalence.FundingBMBWF Austria.

Microbial community analysis via marker gene amplicon sequencing has become a routine method in the field of soil research. In this perspective, we discuss technical challenges and limitations of amplicon sequencing studies in soil and present statistical and experimental approaches that can help addressing the spatio-temporal complexity of soil and the high diversity of organisms therein. We illustrate the impact of compositionality on the interpretation of relative abundance data and discuss effects of sample replication on the statistical power in soil community analysis. Additionally, we argue for the need of increased study reproducibility and data availability, as well as complementary techniques for generating deeper ecological insights into microbial roles and our understanding thereof in soil ecosystems. At this stage, we call upon researchers and specialized soil journals to consider the current state of data analysis, interpretation and availability to improve the rigor of future studies.

Fertilization experiments provide insights into elemental imbalances in soil microbial communities and their consequences for soil nutrient cycling. By addition of selected nutrients, other nutrients become deficient and limiting for soil microorganisms as well as for plants. In this study we focused on microbial nitrogen (N) cycling in a long-term nutrient manipulation experiment. In many soils, the rate-limiting step in N cycling is depolymerization of high-molecular-weight nitrogen compounds (e.g., proteins) to oligomers (e.g., peptides) and monomers (e.g., amino acids) rather than the subsequent steps of mineralization (ammonification) and nitrification. The aim of our study was to determine whether nutrient deficiency directly or indirectly – via changes in plant carbon (C) inputs - affects soil microbial N processing.We collected soil samples from a fertilization experiment, established in 1946 on a hay meadow close to Admont (Styria, Austria). The field experiment consisted of a full factorial combination of inorganic N, P, and K fertilization and a control with no fertilizers. Furthermore, liming (Ca-addition) and organic fertilizer application treatments (solid manure and liquid slurry) were established. In the experiment, plant biomass is harvested three times per year, inducing strong nutrient limitation in plots that have not received nutrient additions (fully deficient or deficient in a single element). We determined gross rates of microbial protein depolymerization, N-mineralization and nitrification via isotope pool dilution assays with 15N-labeled amino acids, NH4+, and NO3-. We hypothesized that N deficiency (lack of N fertilization) would stimulate microbial N mining (depolymerization), and reduce subsequent N mineralization and nitrification. In contrast, we expected that organic fertilization would alleviate microbial C and N limitations, reducing N depolymerization rates and increasing mineralization and nitrification.Our results show that organically fertilized and limed soils have significantly lower gross protein depolymerization rates than plots receiving inorganic N. No significant differences were found comparing gross N-mineralization and gross nitrification rates across the different treatments. Given the higher rates of protein depolymerization in inorganically fertilized soils as compared to organically fertilized and limed soils, microbial N processes seem to be controlled by plant C input and/or soil pH rather than by direct soil nutrient availability. However, depolymerization of macromolecular N does not only supply N to the soil microbial community but also organic C. Thus, the reduced plant C input compared to fully fertilized soils may have caused microorganisms to increase their mining for a C-containing energy source, thereby increasing protein depolymerization rates. In summary, this study suggests that long term nutrient deficiency or nutrient imbalances may affect soil nutrient cycling indirectly by changing plant C inputs (via reduced primary production) and/or changing soil pH, rather than directly, by nutrient availability. This further indicates that soil microbial communities are rather C than nutrient limited.

Arbuscular mycorrhiza (AM) fungi are associated with almost all land plants and provide soil nutrients and other benefits to their plant hosts in exchange for photosynthetic products. While fertilization regimes in managed grasslands or agricultural systems are tailored for increasing plant biomass, their potential effects on AM fungi are rarely taken into account. Nutrient-driven changes in abundance and community composition of AM fungi, however, may feedback on ecosystem performance in the long term. Therefore, it is necessary to get a better understanding on how AM fungal communities respond to changes and imbalances in soil nutrient availabilities.Here, we evaluated how long-term nutrient deficiency of phosphorus (P), nitrogen (N) and potassium (K) affects the abundance and community composition of AM fungi in a mountainous grassland. In addition, we investigated how the responses of AM fungi to those deficiencies were modulated by liming and the type of fertilizer addition (inorganic versus organic).Our study was carried out on a long-term nutrient deficiency experimental grassland site in Admont (Styria, Austria), established in 1946. Different fertilization treatments were applied for more than 70 years in a randomized block design, including numerous combinations of inorganic (P, N, K with/without lime) and organic (solid manure and liquid slurry) fertilizers. The hay meadow at the site is cut three times per year and biomass is not returned to the system. Therefore, biomass and nutrients have been continuously removed for decades, leading to different types of soil nutrient deficiency. In this study, we collected both root and soil samples in July 2019 and quantified AM fungi and other microbial groups by measuring neutral fatty acid (NLFA) and phospholipid fatty acid (PLFA) biomarkers, respectively. Additionally, we applied DNA and RNA-based amplicon sequencing of the 18S rRNA gene to identify AM fungal community composition.Our data shows that deficiencies of one or more elements had a major impact on both AM fungal biomass and community composition. AM fungal biomass was higher in plots that received no fertilizers compared to inorganically fertilized plots, but lower in plots which were deficient only in certain single or multiple elements, specifically in plots fertilized with inorganic N only (i.e., deficient in P and K). Conversely, liming and organic fertilizer amendments increased AM fungal biomass compared to plots containing inorganic fertilizers without lime. Across all treatments, AM fungal biomass was positively correlated with pH and soil water content, and negatively with dissolved N compounds, indicating indirect effects via responses of other soil parameters to nutrient deficiency. Long-term nutrient deficiency also altered plant community composition, which may also have indirectly affected AM fungal communities.We conclude that long-term nutrient deficiency, and in particular the stoichiometry of available nutrients, strongly affects the abundance and community composition of AM fungi in grassland soil. This response may be linked to changes in plant community composition or soil chemistry both as a result and as a cause, emphasizing the complexity of feedbacks determining the response of grassland ecosystems to changing nutrient conditions.

Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding β-galactosidase (β-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the β-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or by immunofluorescent detection using a β-gal-specific antibody. Here, we summarize protocols for the localization of β-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.

International audience; We, the co‐authors of this letter, are an international group of soil scientists at early career stages, from PhD students to postdoctoral researchers, lecturers, and research fellows with permanent positions. Here, we present our collective musings on soil research challenges and opportunities and, in particular, the points raised by Philippe Baveye (Baveye, 2020a, 2020b) and Johan Bouma (Bouma, 2020) on bypass and hyperbole in soil science. Raising awareness about these issues is a first and necessary step. To this end, we would like to thank Philippe Baveye and Johan Bouma for initiating this debate........

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling., SCOPUS: ar.j, info:eu-repo/semantics/published

Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding β-galactosidase (β-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the β-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or by immunofluorescent detection using a β-gal-specific antibody. Here, we summarize protocols for the localization of β-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.

Seagrasses and lucinid bivalves inhabit highly reduced sediments with elevated sulphide concentrations. Lucinids house symbiotic bacteria (Ca. Thiodiazotropha) capable of oxidising sediment sulphide, and their presence in sediments has been proposed to promote seagrass growth by decreasing otherwise phytotoxic sulphide levels. However, vast and productive seagrass meadows are present in ecosystems where lucinids do not occur. Hence, we hypothesised that seagrasses themselves host these sulphur-oxidising Ca. Thiodiazotropha that could aid their survival when lucinids are absent. We analysed newly generated and publicly available 16S rRNA gene sequences from seagrass roots and sediments across 14 seagrass species and 10 countries and found that persistent and colonising seagrasses across the world harbour sulphur-oxidising Ca. Thiodiazotropha, regardless of the presence of lucinids. We used fluorescence in situ hybridisation to visually confirm the presence of Ca. Thiodiazotropha on roots of Halophila ovalis, a colonising seagrass species with wide geographical, water depth range, and sedimentary sulphide concentrations. We provide the first evidence that Ca. Thiodiazotropha are commonly present on seagrass roots, providing another mechanism for seagrasses to alleviate sulphide stress globally.

Context C-type natriuretic peptide (CNP) is critically involved in endochondral bone growth. Variants in the genes encoding CNP or its cyclic guanosine monophosphate (cGMP)-forming receptor (natriuretic peptide receptor-B [NPR-B], gene NPR2) cause monogenic growth disorders. Here we describe a novel gain-of-function variant of NPR-B associated with tall stature and macrodactyly of the great toes (epiphyseal chondrodysplasia, Miura type). Design History and clinical characteristics of 3 family members were collected. NPR2 was selected for sequencing. Skin fibroblasts and transfected HEK-293 cells were used to compare mutant versus wild-type NPR-B activities. Homology modeling was applied to understand the molecular consequences of the variant. Results Mother’s height was +2.77 standard deviation scores (SDS). The heights of her 2 daughters were +1.96 SDS at 7 years and +1.30 SDS at 4 years of age. Skeletal surveys showed macrodactyly of the great toes and pseudo-epiphyses of the mid- and proximal phalanges. Sequencing identified a novel heterozygous variant c.1444_1449delATGCTG in exon 8 of NPR2, predicted to result in deletion of 2 amino acids Met482-Leu483 within the submembrane region of NPR-B. In proband’s skin fibroblasts, basal cGMP levels and CNP-stimulated cGMP production were markedly increased compared with controls. Consistently, assays with transfected HEK-293 cells showed markedly augmented baseline and ligand-dependent activity of mutant NPR-B. Conclusions We report the second activating variant within the intracellular submembrane region of NPR-B resulting in tall stature and macrodactyly. Our functional and modeling studies suggest that this domain plays a critical role in the baseline conformation and ligand-dependent structural rearrangement of NPR-B required for cGMP production.

Axon bifurcation – a specific form of branching of somatosensory axons characterized by the splitting of the growth cone – is mediated by a cGMP-dependent signaling cascade composed of the extracellular ligand CNP (C-type natriuretic peptide), the transmembrane receptor guanylyl cyclase Npr2 (natriuretic peptide receptor 2), and the kinase cGKI (cGMP-dependent protein kinase I). In the absence of any one of these components, the formation of T-shaped axonal branches is impaired in neurons from DRGs (dorsal root ganglia), CSGs (cranial sensory ganglia) and MTNs (mesencephalic trigeminal neurons) in the murine spinal cord or hindbrain. Instead, axons from DRGs or from CSGs extend only either in an ascending or descending direction, while axons from MTNs either elongate within the hindbrain or extend via the trigeminal ganglion to the masseter muscles. Collateral formation from non-bifurcating stem axons is not affected by impaired cGMP signaling. Activation of Npr2 requires both binding of the ligand CNP as well as phosphorylation of serine and threonine residues at the juxtamembrane regions of the receptor. The absence of bifurcation results in an altered shape of termination fields of sensory afferents in the spinal cord and resulted in impaired noxious heat sensation and nociception whereas motor coordination appeared normal.

Enzymes are produced by microorganisms either intracellularly in cell’s cytoplasm and periplasm or extracellularly either as attached to outer surface of cell membranes or released to the soil microhabitats. The distribution of microhabitats in soil is highly heterogeneous with high abundance of microorganisms in the small volume of soil hotspots, e.g., in the rhizosphere - the most important plant-soil interface with very dynamic interactions between roots and microorganisms. Soil zymography is one of the most realistic methods developed to visualize enzyme activity in undisturbed soil at the mesoscale (mm-cm) level using substrate-saturated membranes. However, visualization of enzymatic processes at the micro-scale level remains a challenge. We tested several impregnation strategies of soil sample (e.g., by agarose gel, silicon spray and super transparent silicon mixture) for their suitability for micro-zymography, i.e., for visualization of enzyme activity in undisturbed soil particles at the microscopic level combining fluorogenic substrates with epifluorescence microscopy. The pros- and cons- of various combinations of impregnation and staining of micro-sized soil samples will be discussed.Keywords: enzyme activity, zymography, fluorogenic substrates

Our current understanding of microbial hotspots such as the rhizosphere mainly stems from observations through measurements at the macroscopic scale, integrating a multitude of microbial cells and taxa into a few measured variables. Consequently, we still lack an understanding of the individual participants that actively contribute to processes. Identifying microorganisms and relating their activity to these processes within the soil-plant interface on a microscopic scale represent a missing link in understanding nutrient flux in agriculturally important ecosystems such as rice cultivation.I will present a novel workflow for single-cell isotope imaging in the rhizosphere that combines fluorescence in situ hybridization, gold-targeted secondary electron microscopy, and nano-scale secondary ion mass spectrometry. Based on correlative microscopy and hotspot detection, this approach now allows to (i) identify single bacteria on root surfaces that actively incorporate stable isotopes, (ii) quantify their contribution to processes of interest within a given population, and (iii) potentially trace nutrient fluxes among plants and bacteria on a microscale.Illuminating plant-microorganism interactions on a microscale provides the potential to evaluate the actual impact of bio-inoculants applied as fertilizers and to engineer plant-microorganism associations which may be essential to increase the production of major staple crops for a growing world population.