Applied Soil Ecology

Aims Although ammonia-oxidising archaea (AOA) dominate nitrifier communities in many agricultural soils, the contribution of the less abundant ammonia-oxidising bacteria (AOB) to nitrogen oxide emissions, particularly nitric oxide, remains poorly understood. This study investigated the roles of AOA and AOB in regulating nitric oxide (NO) and nitrous oxide (N2O) emissions from a urea-fertilised grassland soil and evaluated the nitrification inhibitors dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP). Methods A 28-day soil incubation experiment was conducted using a denitrification incubation system to continuously monitor NO and N2O emissions. In parallel, a destructive incubation experiment was performed to determine the dynamics of ammonium (NH-N) and total oxidised nitrogen (TOxN) and to assess changes in the abundance of AOA and AOB following urea application with or without DCD or DMPP. Results AOA accounted for approximately 66% of the total ammonia-oxidising community. Neither inhibitor significantly affected AOA abundance, whereas both suppressed AOB, with DMPP showing stronger and more persistent inhibition than DCD. DMPP reduced NO emissions by 16 to 75%, whereas N2O emissions decreased by only 0.2 to 27%. The greater reduction in NO, together with the preferential suppression of AOB, suggests that low-abundance AOB contributed disproportionately to NO production despite the numerical dominance of AOA. Conclusions These findings suggest that AOB can contribute disproportionately to NO emissions despite their lower abundance relative to AOA. The preferential suppression of AOB by DMPP was associated with a substantial reduction in NO emissions, highlighting AOB-associated processes as a potential target for mitigating reactive N losses from fertilised soils.

Carbon farming practices are increasingly adopted as part of regenerative agriculture to enhance soil carbon sequestration and improve agricultural sustainability. However, their effects on the phyllosphere microbiome, and how these interact with field biodiversity, remain poorly understood. We investigated how field biodiversity and carbon farming practices (cover crops, all-in mixes, adaptive grazing and ley mixture) influence leaf microbial communities across 19 Finnish farms using ITS and 16S amplicon sequencing. Foliar fungal - but not bacterial - diversity increased with plant species richness. Carbon farming altered both fungal and bacterial community composition, but only fungal community was positively associated with disease load. Network analysis identified three candidate keystone taxa: Alternaria sp. (FASV 34) and Sporobolomyces roseus (FASV 2070), which were mutually exclusive, and Methylobacterium sp. (BASV 75), which co-occurred with FASV 2070. Together, these findings demonstrate that different carbon farming practices shape distinct leaf microbiomes cover crop systems tending to favor potential fungal pathogens, whereas ley mixture and adaptive grazing strategies promote microbial taxa associated with healthier phyllosphere communities. Our study underscores the importance of understanding the complex microbial interactions within leaf ecosystems to inform sustainable agricultural practices.

Soil nematodes play key roles in regulating organic matter decomposition, nutrient cycling and trophic interactions in the soil ecosystem, and are very useful bioindicators of soil condition and soil health. But to characterise them, we need reliable measurements of nematode biodiversity in soils. DNA-based techniques such as metabarcoding have recently explored their biodiversity, with successful results. However, there are many methodological steps that can modify the outcome considerably and require informed decisions. In this study, we tested the effect on nematode alpha and beta diversity metrics of three methodological steps, namely i) type of extraction of genetic material from the matrix, either from soil directly with different starting amount of material or extracting nematodes first via the Baermann or Oostenbrink methods, ii) the choice of taxonomically-relevant DNA segment for amplification, with a nematode specific primer (NemF/18Sr2b) and a universal eukaryotic primer (3NDf/1132rmod), both within 18S rDNA, and iii) the selection of appropriate reference library for taxonomic assignment (comparing three nematode specialised databases and three broad taxonomic databases). Observed differences in alpha diversity were more pronounced when comparing the nematode extraction vs soil extractions. In particular, direct soil extractions co-extracted many non-nematode taxa, especially if amplified with a universal primer, which can significantly reduce the number of nematode reads obtained and therefore the accuracy of nematode community indexes and other nematode-based metrics. The choice of the reference database strongly affected the taxonomic identification, which calls for extreme caution when making this selection. Notably, specialised databases tended to over assign reads to nematodes that were detected as non-nematodes by broad taxonomic databases. However, beta diversity metrics were consistent across methods, highlighting these metrics as robust against methodological choices. Our study clearly demonstrates that the choices of methods for nematode biodiversity studies using molecular tools can have a big impact on the results, and decisions should be made consciously and carefully.

Much of the persistent soil organic carbon (SOC) pool is microbial in origin: microorganisms process plant carbon into biomass and their necromass can associate with mineral surfaces, contributing to long-term carbon persistence. This has generated interest in microbial interventions such as inoculation to restore carbon in degraded croplands, yet their efficacy remains uncertain. The uncertainty reflects a more fundamental, unresolved question: are microbially-mediated carbon transformations governed principally by the composition of the microbial community or by the soil microenvironment? To disentangle these drivers, we conducted a reciprocal microbial community transplant experiment, introducing communities of contrasting origin from locally adjacent grassland and cropland soils into sterilised grassland and cropland soils, and incubating them for eight months with regular organic inputs. This design decouples the inoculum from the microenvironment, allowing their individual and interactive contributions to be quantified. Fungal assembly was influenced more by the inoculum, consistent with dispersal limitation, whereas bacterial assembly was governed more by the microenvironment, consistent with environmental selection. Despite receiving the same organic carbon inputs, grassland and cropland recipient soils showed distinct SOC trajectories, indicating that the soil microenvironment strongly constrained net carbon retention. Within this constraint, community composition also mattered: introducing grassland rather than cropland communities increased fungal diversity and fungal necromass and led to better SOC outcomes, expressed as net gain or reduced loss. These results show that SOC accrual emerges from interactions between the soil microenvironment and microbial community composition, with fungal community assembly particularly associated with necromass accumulation and carbon retention. They highlight the need to consider both soil conditions and microbial community composition when developing strategies to enhance SOC accrual.

Globally, contaminated soils pose serious risks to agriculture, biodiversity of ecosystems, and human health. Although a variety of monitoring and remediation methods have been developed over the years, there remains a lack of theoretical modelling to better understand the impact of contamination on soil carbon in a microbially-mediated system. In this study, we develop a model which incorporates contaminated soil organic carbon, uncontaminated soil organic carbon, dissolved organic carbon, microbes, extracellular enzymes, and CO2, to explore how different amounts of contamination and modes of contamination (pulse and press) affect microbially-mediated carbon flux within both vegetated and barren environments. The model represents contamination as a pool of contaminated soil organic carbon that is more resistant to microbial degradation than uncontaminated soil organic carbon. The results show that, in barren environments, the amount and type of contamination has no notable impact on the soil system. In contrast to this, in vegetated environments, plants mitigate the impact of large contamination inputs with differences in outcome depending on the amount and type of contamination.

Urbanisation is widely recognised as a major driver of environmental change, yet its impacts on soil microbiomes remain poorly understood, particularly outside megacities and beyond bacterial taxa. Population density is frequently used as a proxy for urban intensity, but its relevance across different microbial kingdoms and urban contexts is unclear. Here, we investigated how soil microbial communities vary across population density bands in the regional, temperate city of Hobart, Australia. Using a multi-kingdom metabarcoding approach, we analysed bacterial, fungal, and protozoan communities from soils collected in urban green spaces along a population density gradient. Bacterial alpha-diversity differed significantly across population density bands, exhibiting an inverse U-shaped relationship, with the highest diversity at green spaces in intermediate population density areas. In contrast, fungal and protozoan alpha-diversity did not vary with population density. Community composition (beta-diversity) did not differ consistently across density bands for any microbial group, and variation in soil physicochemical properties explained more community variation than population density alone. These results indicate that population density is a selective, rather than universal, driver of urban soil microbiomes and that responses to urbanisation differ markedly among microbial kingdoms. Our findings highlight important limitations of using population density as a sole indicator of urban impact and challenge the generalisability of conclusions drawn from bacteria-focused studies in large cities. By demonstrating kingdom-specific and context-dependent responses in a regional city, this study underscores the need for multi-kingdom, multi-scale approaches to better understand how urbanisation shapes soil microbiomes and their potential implications for urban ecosystem functioning and human health.

Characterizing the enzymatic accommodation of circular agricultural fertilizers is critical for informing strategies to remediate widespread soil sulfur hunger. Here, we present an exploratory in-silico investigation evaluating the active-site cleft of sulfate thiohydrolase (SoxB) across representative soil Proteobacteria. Following the crystallographic precedent of uncomplexed thiosulfate in PDB 2WDE, site-directed molecular docking indicated that the free thiosulfate polyanion binds favorably within the catalytic pocket (predicted affinity: -3.506 kcal/mol), yielding a substantially lower empirical energy barrier than hydrophobic elemental sulfur (S8, -1.409 kcal/mol). Comparative evaluation against the alkaline-adapted Thiobacillus denitrificans homolog revealed an elevated predicted binding affinity of -4.610 kcal/mol, suggesting a potential structural accommodation in high-pH calcareous soils (pH > 8.0). Unrestrained 5.0 ns all-atom molecular dynamics in explicit TIP3P solvent demonstrated initial structural stability of the unliganded host backbone (RMSD = 1.10 +/- 0.15 Angstrom). Energetic decomposition indicated that ligand association is governed predominantly by electrostatic interactions (delta-E_elec ~ -40 kcal/mol) with basic residues (His146, His269, Trp147; RMSF < 0.60 Angstrom), with a single-trajectory unbinding event observed at 3.2 ns. These computational observations provide an exploratory baseline characterizing the active-site electrostatic landscape of SoxB, generating working hypotheses for downstream empirical soil microcosm and in-planta trials.

Abstract Background: Soil microbiomes play critical roles in plant health and resilience, yet it is still poorly understood how disruptions in local microbial communities, such as during soil management, impact these ecological functions. In this study, we investigated how compost addition, a widely used soil management practice, and microbiome origin influence microbiome-mediated plant resistance to insects. We combined surveys of working organic farms across New York with controlled laboratory experiments, amplicon sequencing, plant-insect bioassays, and mass spectrometry. Results: Compost use was associated with shifts in the relative abundance of 20 fungal and 2 bacterial genera across ten working farms. However, no consistent changes in bacterial or fungal diversity or community structure in fields with compost were observed across the farms. Under controlled conditions using a common compost source, compost increased rhizosphere microbial diversity and significantly altered rhizosphere community structure, including the relative abundance of 6 fungal and 41 bacterial genera. Despite these substantial community-level changes, compost did not affect populations of green peach aphids (Myzus persicae) on kale (Brassica oleracea). Instead, aphid populations and plant salicylic acid concentrations were more strongly associated with microbiome origin, demonstrating that compost can substantially restructure soil and rhizosphere microbial communities without corresponding changes in microbiome-mediated plant resistance to insects. Conclusions: Our findings demonstrate that microbial diversity and compositional responses to management are not necessarily reliable indicators of all microbiome functions. More broadly, this work highlights the importance of local soil microbiome variation in shaping plant-insect interactions and emphasizes the need to link community-level responses to direct functional measurements when evaluating microbiome responses to agricultural management.

The mechanisms by which coarse woody debris (CWD) affects soil microbial diversity across different spatial scales and forest disturbance regimes remain poorly understood. This study investigated the impact of CWD from multiple tree species on forest floor bacterial and fungal communities under varying landscape-scale conditions, specifically comparing old-growth forests situated on Phaozems and Retisols with mass-windthrow areas located on loamy Retisols and sandy Podzols. We analyzed both chemical compound and metagenomic profiles of 63 CWD-associated soil samples collected from the Kaluzhskie Zaseki Nature Reserve (European part of Russia). High-throughput Illumina sequencing of the 16S rRNA gene and the ITS region was used to profile both bacterial and fungal communities, respectively. Scale-dependent diversity patterns and taxonomic accumulation were assessed using GLM, NMDS, dbRDA, and iNEXT frameworks. Our findings revealed a profound spatial decoupling in forest floor microbial communities, where a localized reduction in alpha-diversity under CWD drove a sharp increase in landscape-scale gamma-diversity. While standard soil physicochemical properties rigidly dictated baseline matrix richness, CWD operated as a powerful micro-environmental hotspot overriding these constraints. Deadwood inputs filtered out dominant matrix-associated taxa, creating highly specialized micro-refugia characterized by high compositional dispersion. This niche selection was particularly pronounced beneath Quercus logs on Phaozems and Retisols. Furthermore, post-windthrow microbial responses were heavily filtered by wood traits and baseline soil textures. On loamy Retisols within the windthrow mosaic, this breakaway dynamic was highly taxon-specific: while Acer and Fraxinus CWD triggered partitioned, site-specific hotspot dynamics yielding an exceptionally large pool of unique ASVs for bacteria, the distinct enrichment for fungal communities was primarily driven by Ulmus CWD and log-free windthrow areas. Ultimately, iNEXT accumulation profiles confirmed that deadwood does not merely multiply common soil taxa, but enriches the forest landscape by injecting critical beta-level heterogeneity into the soil matrix. These insights emphasize the role of diverse CWD retention in buffering forest microbiomes against large-scale environmental disturbances across heterogeneous soil landscapes.

Soil microbes regulate whether carbon is retained in soils or returned to the atmosphere through respiration, but the extent to which microbial community characteristics improve the prediction of heterotrophic soil CO2 production beyond predictions by environmental controls remains unclear. We tested this across a topographically structured forest-to-marsh gradient in coastal Oregon by measuring heterotrophic soil respiration, soil physicochemical properties, PLFA-based microbial biomass, metagenomic taxonomic composition, and functional gene-based trait indicators. Across the gradient, soil moisture increased from forest to marsh, while mineral soil and organic matter C:N decreased. These environmental shifts were accompanied by strong but uneven microbial responses: total microbial, fungal, and bacterial biomass declined from forest to marsh, taxonomic composition showed the strongest structuring by environmental conditions, and functional gene-based indicators showed mixed relationships with the gradient. Environmental model comparisons identified soil moisture and organic layer C as the strongest baseline predictors of respiration. Among microbial descriptors, only a small subset improved respiration prediction beyond this environmental baseline. The fungal:bacterial (F:B) biomass ratio produced the largest increase in model fit and the greatest reduction in AICc, whereas the best taxonomic and functional-gene predictors yielded more minor gains. Our results showed that the microbial descriptors most responsive to environmental gradients were not the ones most useful for predicting soil respiration; instead, a relatively simple biomass-partitioning metric captured respiration-relevant microbial variation more effectively than finer taxonomic and genomic descriptors. This suggests that F:B ratio may be especially useful for representing respiration-relevant microbial variation in local landscape-scale studies and for future carbon cycle models applied across heterogeneous ecosystem transition zones.

As circular agricultural inputs are increasingly sought, feather hydrolysate (FH), derived from keratin-rich poultry waste, has emerged as a promising fertilizer and biostimulant. However, how FH shapes soil microbiome succession over time, and what functional potential this restructuring carries, remains poorly understood. Here, we used multi-marker high-throughput sequencing of 16S, 18S, and ITS rDNA to track soil microbial communities over 30 days following FH amendment, with sampling at days 0, 1, 4, 14, and 30. FH induced a rapid and reproducible microbial succession characterized by an early decline in alpha diversity and a pronounced day-1 bloom of copiotrophic taxa, as inferred from predicted rrn operon copy-number profiles. This initial response was accompanied by enrichment of bacterial genera previously associated with plant growth-promoting traits, including pathogen suppression, nutrient solubilization, siderophore production, phytohormone synthesis, and ACC deaminase activity. Although these groups gradually declined after the initial peak, several remained at significantly elevated abundances throughout the 30-day experimental period. In parallel, FH-treated soils showed a sustained reduction in the cumulative relative abundance of genera annotated as putative phytopathogens. By integrating temporal community profiling, rrn-based life-strategy inference, and literature-derived functional annotation, we show that FH acts as a strong ecological filter, shifting the soil microbiome toward copiotrophic and putatively plant-beneficial taxa. These findings provide a temporal framework for understanding FH-mediated microbiome restructuring and highlight the need for future studies linking inferred microbial functional potential to measured disease incidence, nutrient fluxes, and plant performance across diverse soil contexts.

Soil is one of the most biodiverse habitats on the planet, yet the extent of soil biodiversity knowledge in Canada is unknown. To better understand our knowledge, we systematically mapped soil invertebrate biodiversity knowledge from peer-reviewed literature to identify spatial and taxonomic gaps and to assess whether areas projected to experience different magnitudes of climate change are adequately sampled. A total of 1,176 Canadian sampling locations were identified from 427 articles. We found biases in sampling coverage spatially and in relation to current and projected climatic conditions. The geographic region above 60 degrees North is sparsely sampled. Colder regions (< -0.12 C) and regions receiving the least (< 646.47 mm), moderately high (2268.67 to 2809.40 mm), and high (2809.40 to 3350.14 mm) amounts of precipitation were under sampled. Regions projected to have the smallest (< 0.86 C) and greatest (> 1.59 C) magnitudes in temperature change and regions projected to have moderate (-2.06 to +2.69 mm) to large changes (+2.69 to +7.44 mm) in precipitation were underrepresented. The sampled locations also suffer from taxonomic biases which were skewed toward Nematoda (19%), Annelida (18%), Acari (15%), and Collembola (14%). Our results suggest that methods such as stratified sampling is essential to capture underrepresented regions, taxonomic groups, and an array of current and projected climatic conditions. Additionally, shifting to a multispecies sampling approach is essential for broader soil ecosystem knowledge. Importantly, data from well-sampled areas should be examined to determine how it can be leveraged to advance conservation management of soil invertebrates.

The potential of alternative agricultural systems, such as organic or conservation agriculture, for biodiversity restoration has been widely investigated. However, variability of practices within each of these two categories of agricultural system has rarely been accounted for. Furthermore, these systems have most often been studied separately, isolating either organic or conservation agriculture (itself often reduced to a soil tillage reduction), and rarely analyzing these systems side by side. This study attempts to disentangle these multiple interrelationships by sampling carabid beetles in a network of 48 commercial Belgian farms including organic agriculture (OA), conservation agriculture (CA), farms combining the two approaches (OCA) and farms applying conventional practices (CVA). In each farm, we sampled carabids using pitfall and emergence traps in one winter cereal field and collected a detailed description of crop management practices. We found that community composition was primarily structured by seasonality early in the season (March, April) followed by convergence (May, June) driven by the aggregation of the most dominant species. Based on agroecosystem classification, organic certification (i.e., the absence of synthetic pesticide use) exerted a twofold stronger effect than application of conservation agriculture principles (i.e., reduced or no soil tillage) on carabid beetle community composition. Notably, OCA supported carabid assemblages that were structurally distinct from those of CA and CVA. Furthermore, carabid communities associated with OA differed significantly in composition from those of CVA. Observed and estimated species richness were also significantly higher in OCA than in CA and CVA, while OA differed from CVA only for observed richness. Beyond differences among agricultural systems, community responses were associated with specific management practices, which explained nearly a quarter of the compositional variation with significant influence of the duration since organic conversion and the frequency of synthetic pesticide application. Carabid abundances were negatively affected by synthetic pesticides and moldboard ploughing, whereas richness was more strongly related to the time since conversion to OA, cover crops, and organic amendments. Overall, these results position synthetic inputs and time since conversion as the main filters over carabid communities. Our study highlights the importance of jointly considering system level analyses and detailed management practices to better understand the complex relationships between agricultural practices and carabid communities.

Reforestation is a leading natural climate solution, but bulk soil organic carbon (C) often responds slowly, obscuring early belowground change. We tested whether soil aggregates reveal early structural reorganization that affects C retention pathways at a three-year-old experimental reforestation planting established on former pasture in Oregon, USA. We sampled soils at 0-20 and 20-40 cm beneath incense cedar (Calocedrus decurrens; arbuscular mycorrhizal [AM]), black cottonwood (Populus trichocarpa; AM and ectomycorrhizal [EcM]), ponderosa pine (Pinus ponderosa; EcM), and treeless controls. We measured bulk soil C concentration and C:N, aggregate size distribution and mean weight diameter (MWD), fraction-associated C, and, in a subset of surface aggregate fractions, natural-abundance {delta}13C to evaluate soil C pools, physical structure, C distribution, and C processing. After three years, reforestation did not produce significant differences in bulk soil C between planted trees and treeless controls. In surface soil, MWD averaged 36% higher under incense cedar and 58% higher under black cottonwood than under controls, whereas ponderosa pine remained similar to controls. The clearest treatment differences in C distribution occurred in macroaggregates. Incense cedar and black cottonwood had higher large (>2000 m) macroaggregate-associated C than controls, while black cottonwood combined a greater proportion of large macroaggregates with lower C concentrations, indicating that structural development and C accumulation were partly decoupled. Species patterns were broadly consistent with stronger early aggregate responses under AM-compatible species in former-pasture legacy conditions; by contrast, the EcM-associated ponderosa pine remained closer to treeless controls across multiple aggregate measures. Treatment effects weakened with depth and were limited in microaggregates (250-53 m) and silt-and-clay (<53 m) fractions. Smaller aggregate fractions were progressively enriched in {delta}13C, consistent with greater C processing in protected fractions, but treatment-level isotope differences had not yet emerged. Soil aggregates showed species-associated belowground reorganization, revealing how tree identity and former-pasture legacy may shape early soil C organization before consistent differences emerged in bulk soil C. Aggregate measures can complement bulk soil C measurements in restoration monitoring as early indicators of belowground soil C trajectories shaped by tree species identity and land-use legacy.

Deadwood is a common ephemeral resource in boreal forests, but arthropod succession across the full decay gradient and transition into soil remains poorly understood. We analyzed the successional trajectory of arthropods in deadwood to soil. Further, we investigated whether the succession pattern differed in previously clear-cut and near-natural forests in southeastern Norway. This was done by DNA metabarcoding analyses of arthropod communities in collected deadwood and soil samples. We detected 741 arthropod OTUs, with 565 OTUs in deadwood and 291 in organic soil layers, of which 115 were found in both deadwood and organic soil layers. Arthropod richness increased with deadwood decay stage and organic soil layers supported higher richness than all deadwood decay stages. Community composition of arthropods changed significantly along the decay gradient, with late-decay stages sharing more OTUs with organic soil layers than with earlier decay stages. This suggests a gradual transition between communities in highly decayed deadwood and organic soil layers. Forest type (previously clear-cut vs. near-natural forest) had limited effects on overall richness and community composition, although previously clear-cut and near-natural forests supported partially distinct species pools and differed in co-occurrence patterns in late decay and organic soil layers. Our results show that arthropod communities are strongly structured along the decay gradient from freshly dead wood to organic soil layers. This highlights the importance of retaining deadwood across different decay stages for maintaining arthropod diversity and community convergence between deadwood and soil in boreal forests.

Recent years have witnessed the discovery of an immense diversity of protists in almost all ecosystems on Earth. However, a major challenge consists in identifying diversity patterns and the drivers that explain the distribution of this diversity. While several works have focused on local parameters, only a few studies have addressed this question at regional scales. In this study, we characterize the diversity and distribution of Arcellinida (testate amoebae) at regional scale of a climatically contrasted country, Bulgaria. We collected 75 samples geographically evenly distributed and representative of the country's natural ecosystems and used metabarcoding to determine their diversity. Communities composition differed markedly among habitats, revealing sharp beta diversity patterns. "Mesophilic" environments such as forests and ponds were most diverse, while acidic and oligotrophic Sphagnum peatlands were poorest and hosted the highest proportion of unique taxa. Our analyses of richness-environment based on a Random Forest approach applied to all soil systems indicated that stable humidity (low silt content, low precipitation seasonality) and intermediate soil carbon stocks had a positive effect on richness. The predicted increase in continentality in the next decades in Bulgaria calls for testing climatic models to evaluate the potential risk of declining Arcellinida biodiversity.

This study developed a streamlined, sensitive and rapid method for co-detection of low concentrations of Salmonella enterica, Shigella flexneri, Listeria monocytogenes, and pathogenic Escherichia coli from soil. First, autoclaved soil from Iowa with serial dilutions of bacteria to confirm individual species recovery rates on selective agar by qPCR under varying primary enrichment times was used. All pathogens were detected by qPCR of colonies recovered from replicate sterile soils spiked with 10^1 or greater bacteria within 5 hours of enrichment. Shiga toxin expressing E. coli (STEC) and L. monocytogenes were consistently co-recovered in autoclaved and natural soil, pathogen spiking concentration, and secondary enrichment time. S.enterica was consistently recovered at autoclaved and natural concentrations, while S. flexneri detection rates fluctuated. Twenty-three soil samples from Kenyan neighborhoods, with prior evidence of contamination, was compared to qPCR detection at each protocol stage: processing, primary enrichment, secondary enrichment, and selective colony isolation. Detection rates substantially increased for S. enterica, STEC and other pathogenic E. coli after enrichment, with optimal detection rates occurring after secondary enrichment. Testing colonies from selective agar resulted in high false negative rates, possibly reflecting competition from native soil microbiota that was absent in spiked sterile soil experiment. Evaluation of the method with other environmental sample types yielded consistent detection patterns. Evidence suggests a protocol including 24-hour primary enrichment, followed by 24-hour secondary enrichment, and qPCR testing of soil DNA at primary and secondary steps is a reproducible and more expedient method for co-detecting common Enterobacteriaceae and L. monocytogenes pathogens in soil.

1. Grassland ecosystems are pivotal for global biodiversity, carbon (C) storage and food security. This biome, however, together with the ecosystem services it provides, is threatened by global change perturbations. These perturbations rarely occur in isolation and most often affect environments simultaneously, which is a pressing issue in ecology. Nevertheless, most of the research conducted so far has focussed on single simultaneous perturbations or pairs thereof. The few examples considering the effect of more than two simultaneous perturbations focussed only on plants or soil separately, which ignores the potential buffering or feedback effects on ecosystem functioning induced by plant-soils interactions. 2. To tackle this knowledge gap, we used an experimental setup involving mesocosms (soil and plants intact) collected in an extensively managed permanent grassland. Mesocosms were arranged in a greenhouse experiment where we applied individual and combined global change perturbations (up to ten in total) including application of fertilisers, pesticides and antibiotics, simulated trampling and grazing, heat stress and drought. Measured ecosystem functions and properties included a large set of biological, physical and chemical soil and plant associated variables. 3. Results showed that multiple perturbations in most cases had negative effects irrespective of the direction found for individual perturbations. However, in contrast to previous bare soil experiments, we only found scarce evidence for substantial synergistic effects of simultaneous perturbations in our experiment conducted with mesocosms that contained vegetation. This suggests a mitigating impact of plants on soil responses to global change. Moreover, we found a strong dominative effect of drought on ecosystem functioning, driving most of the effects observed. 4. Synthesis: Our results highlight the importance of evaluating global change impacts under more realistic settings, i.e., applying multiple perturbations simultaneously and including plant-soil interactions. Mitigation of global change in terrestrial ecosystems will only be possible once we holistically understand how ecosystems respond to altered conditions.

1. Plant roots employ multiple strategies to acquire soil resources, including the outgrowth of root hairs and the association with arbuscular mycorrhizal fungi (AMF). These structures represent alternative pathways to access soil resources. Evidence is derived mostly from studies within single species or multi-species experiments under controlled conditions. It remains unclear whether this trade-off holds under natural conditions and whether it manifests consistently across different scales of biological organization. 2. We analysed 81 grassland species representing grasses, forbs and legumes, sampled under heterogeneous field conditions in three regions across Germany, allowing for analyses (i) across different species (interspecific), (ii) across communities (intercommunity), (iii) within plant species (intraspecific) and (iv) within root systems of individual plants (intra-individual). Fine root traits related to AMF colonization (including scoring of fungal hyphae and arbuscules) and root hair growth (including length and incidence) were quantified. 3. Comparing plant species, we observed a strong negative correlation between mycorrhizal colonization and root hair traits, confirming previous studies working under controlled conditions. The negative relationship between root hair length and mycorrhization was also evident when comparing mean trait values at the plant community scale. Among different individuals of the same species (i.e. intraspecific scale), there was no consistent pattern of trait-trait relationship. When comparing within roots of the same plant (i.e. intra-individual scale), co-occurrence of root hairs and mycorrhiza was less likely than what would be expected by chance, with forbs showing the most consistent negative pattern. 4. Our study demonstrates that the hypothesized trade-off between AMF and root hairs is a robust pattern under field conditions at the community and interspecific scale. At the intraspecific scale, no uniform trade-off pattern can be discerned, and within single root systems the negative trade-off pattern is widespread but weak. This indicates that ecological and evolutionary constraints shape trait-trait relationships at higher scale, whereas physiological limits do not restrict trait co-occurrence at lower scales.

The Peruvian Andes, and especially its slopes towards the Amazonian Basin are well-known biodiversity hotspots for plants and animals, which represent the visible part of biodiversity. However, not much is known about microeukaryotic diversity, despite the significant role protists play in the ecosystem. Based on a molecular survey of the diversity of Arcellinida (a group of amoebae well known for its environmental sensitivity and restricted geographical distributions), we estimated its diversity along an elevation gradient spanning more than 3000 meters. Surprisingly, Neotropical forest Arcellinida were less abundant and diverse than in most other extratropical biomes; nevertheless, communities differed strongly in their taxonomic composition as compared with forests in higher latitudes. Notably, sequences related to Trigonopyxis were most abundant, whereas this group occurs only marginally in extratropical settings. Elevation influenced beta diversity, showing different assembly processes occurring at different elevation belts. Canopy cover positively influenced both phylogenetic and taxonomic diversity, showing the importance of above and below ground integration in tropical forests.