Abstract. Soil health assessment increasingly relies on biological indicators because of their sensitivity and direct links to ecosystem functioning. However, conventional laboratory methods are time-consuming, require specialized infrastructure, and are often incompatible with rapid decision-making in applied contexts. Several rapid or field-deployable tools have recently been developed to address this limitation, but their comparability with standard laboratory methods remains insufficiently evaluated. Here, we compared four rapid approaches with their corresponding laboratory reference methods in a long-term grassland experiment: aggregate stability (SLAKES), soil respiration (portable CO2 analyzer), microbial biomass carbon and fungal-to-bacterial ratio (microBIOMETER®), and enzyme activities (Soil Enzymatic Activity Reader, SEAR). Agreement between methods was assessed using Spearman correlations, redundancy analyses and Procrustes analysis. Aggregate stability showed strong correspondence between rapid and laboratory measurements (R = 0.64), whereas soil respiration exhibited weak agreement, likely reflecting that in situ and laboratory approaches capture different aspects of respiratory activity. Microbial biomass carbon displayed moderate comparability between methods (R = 0.51), while fungal-to-bacterial ratios did not. Enzyme activities measured with SEAR were generally consistent with laboratory assays. Multivariate analyses indicated that overall, rapid methods captured ecological patterns similar to those revealed by laboratory protocols. These findings support the use of selected rapid tools as complementary or alternative options when laboratory facilities are unavailable or timely soil health information is required to inform management decisions.

Agricultural practices can be main drivers of antibiotic resistomes in agroecosystems. The application of manure-based fertilizers, the use of biocides, and the entry of heavy metals to agricultural soils associated with fungicide and/or manure application can all influence the prevalence and dissemination of antibiotic resistance genes (ARGs) in agricultural settings. Alternative soil amendments, such as biochar, have been proposed for the mitigation of antibiotic resistance in agriculture. Here, we investigated the effects of the following treatments, individually and in combination, on soil and lettuce resistomes: (i) mineral (NPK) vs. organic (aged cow manure, non-spiked or spiked with two doses of oxytetracycline) fertilization; (ii) copper application; and (iii) biochar application. We assessed their impact on soil physicochemical and microbial properties, including prokaryotic community composition, as well as the relative abundances of ARGs and MGE-linked genes in soil and lettuce plants. The application of mineral fertilizer, oxytetracycline-spiked manure, and biochar increased the abundance of ARGs and MGE-linked genes in soil by 2- to 16-fold, along with increases in some prokaryotic families linked to ARG-harboring MGEs. In contrast, copper application reduced soil microbial activity but did not affect the soil resistome. Our findings underscore the importance of evaluating the impact of agricultural practices on soil and crop resistomes, with fertilization emerging as the practice with the greatest impact under the conditions of this study. Such assessments are critical for proposing management strategies (e.g., pre-treatment of manure-based amendments) aimed at mitigating the transfer of ARGs to potential bacterial human pathogens.

The exponential growth of genomic data has created a pressing need for methods capable of interpreting complex biological information, especially in fields like paleogenomics and environmental metagenomics. Ancient DNA (aDNA) analysis faces challenges such as degradation and contamination, while soil metagenomic DNA (soDNA) analysis is hindered by microbial diversity and incomplete reference databases. To address these limitations, this study proposes the polymerase chain reaction (PCR).Perturbation Theory and Machine Learning (PTML) methodology, which integrates machine learning with Perturbation Theory to analyze genetic sequences without the need for alignment. Two models are developed: the first classifies bacterial aDNA sequences extracted from Miocene amber; the second predicts tree health using microbial gene abundance in forest soils. Both rely on entropy‐based descriptors (θk) and structural differences (Δθk) between query and reference sequences, which serve as perturbation operators for supervised learning algorithms. This approach allows the detection of meaningful patterns even without complete genomic references. The aDNA model achieves 99.65% sensitivity and 99.81% specificity, while the soDNA model reaches 98.85% sensitivity and 92.56% specificity. These results confirm the robustness and applicability of PCR.PTML in diverse genomic contexts, presenting it as a valuable tool for ancient DNA classification and environmental metagenomics analysis.

Meeting future food demands while preserving ecosystem integrity requires agricultural systems that sustain soil biodiversity. Despite its critical role in regulating soil fertility, crop resilience, and food production, the response of soil biodiversity to sustainable agricultural practices remains poorly understood, particularly across multiple groups of organisms. Here, we evaluated the effects of organic amendments, cropping pattern diversification, and microbial inoculation on soil biodiversity using 23 treatment-control pairs from 11 case studies across six Mediterranean countries and three different cropping systems. We employed an integrative methodology combining multitargeted DNA metabarcoding (16S rRNA, ITS, 18S rRNA, COI, Oligo01) and morphological identification (Macfadyen extractor and pitfall traps) to study the structural and functional diversity of soil microorganisms, microfauna, microarthropods, and macrofauna. Data from molecular methods differed between primers and from morphological data; specifically, 18S rRNA and COI yielded markedly different diversity estimates, and both underperformed morphological identification for soil fauna resolution, indicating the need for primer standardisation, increased soil input mass and methodological complementarity between molecular and morphological approaches. Overall, our results show that compost addition positively influenced prokaryotic chemoheterotrophs, epigeic Collembola, spider richness, and total macrofauna abundance, with mulching eliciting broader positive responses. Cropping pattern diversification (i.e., rotations, cover crops, intercropping) more frequently enhanced than reduced soil biodiversity metrics, with 32% of effect sizes being moderately positive compared to 24% moderately negative. Microbial inoculations had limited influence on biodiversity, consistent with their intended role of enhancing specific functions while minimising disruption to resident communities. Although positive responses to sustainable agricultural practices were more frequent than negative ones, effects were often not statistically significant and were taxon- and context-dependent, highlighting the importance of site-specific assessments.

Antibiotic resistance is a major global health concern. Understanding how climate change-related factors can influence antimicrobial resistance patterns in the environment is essential for developing efficient One Health-based resistance control strategies. In this study, under field conditions, we assessed the combined effects of soil warming and biocide (herbicide, fungicide, insecticide) application on soil physicochemical and microbial parameters, potato yield, and the soil resistome using the targeted metagenomics ResCap platform to quantify antimicrobial resistance genes (i.e., antibiotic resistance genes, metal resistance genes, biocide resistance genes, relaxase genes). To our knowledge, this is the first field study specifically designed to investigate the effects of the interaction between agricultural practices (i.e., biocide application) and soil temperature on the environmental resistome, thereby providing insight into the potential contribution of agroecosystems to the reported link between rising air temperatures and the increasing prevalence of antibiotic resistance. Our results indicate that neither soil warming nor biocide application were key factors affecting the observed variation in the abundance of resistance genes. Instead, the temporal dynamics during the growing season, as reflected by the observed differences between soil sampling times, was the variable with the strongest effect on soil resistome dynamics. These findings highlight the importance of studying the temporal dynamics during the growing season when evaluating how agricultural practices and climate change-related factors can influence the environmental resistome.

Antibiotic resistance is a growing global problem, with agricultural practices and climate change as substantial contributors to the spread of antibiotic resistance genes (ARGs) in the environment. We investigated the effect of drought and fertilization type (organic vs. mineral) on radish crop growth and soil prokaryotic communities, with special emphasis on the radish and soil resistomes, as measured by the relative abundance of ARGs and mobile genetic element (MGE)-linked genes. Manure fertilization significantly increased ARG relative abundances in soil, compared to mineral fertilization. Drought and the presence of radish plants emerged as key variables regulating the association between ARGs and MGE-linked genes. Nonetheless, despite radish being a belowground crop, no direct connection was observed between the soil and crop resistomes. These results suggest that soil moisture and fertilization strategies do not necessarily increase the risk of ARG transfer to human pathogens through crop consumption. Consequently, a robust risk assessment of the environmental resistome must account for all compartments within the transmission chain. Together, our findings highlight the complex interplay between agricultural practices and climatic factors in shaping the soil and crop resistome.

This article presents a dataset of antibiotic resistance gene abundances obtained when exposing soil, previously amended with oxytetracycline-spiked cow manure, to different temperatures and moisture contents as two highly relevant climate change-related variables. The absolute abundances of six antibiotic resistance genes (ARGs) and two mobile genetic element (MGE)-linked genes were determined by droplet-digital PCR. Data on soil microbial biomass carbon, the total abundance of the 16S rRNA gene, and basal respiration are also included to show the effect of the climate change-related variables on the biomass and activity of soil microbial communities. The dataset presented in this article contains raw observations (including the soil´s physicochemical characterization), as well as analysis-derived data, on the effects of climate change-related variables on the risk of antibiotic resistance occurrence and spread in soils amended with animal manure, a topic of the utmost importance given the potential links between the environmental resistome and the human resistome. The data provided in this article are of much interest to researchers dealing with the potential impact of agricultural practices (i.e., organic fertilization) on antibiotic resistance under the current scenario of climate change.

Antibiotic resistance is a growing global problem, with agricultural practices and climate change as substantial contributors to the spread of antibiotic resistance genes (ARGs) in the environment. We investigated the effect of drought and fertilization type (organic vs. mineral) on radish crop growth and soil prokaryotic communities, with special emphasis on the radish and soil resistomes, as measured by the relative abundance of ARGs and mobile genetic element (MGE)-linked genes. Manure fertilization significantly increased ARG relative abundances in soil, compared to mineral fertilization. Drought and the presence of radish plants emerged as key variables regulating the association between ARGs and MGE-linked genes. Nonetheless, no connection was observed between the soil and crop resistome, despite radish being a belowground product, suggesting that, under our experimental conditions, the consumption of a belowground crop product does not pose a potential risk of transmission of ARGs from agroecosystems to human bacterial pathogens. Our findings highlight the complex interplay between agricultural practices and climatic factors in shaping the soil and crop resistome.

Soil organic matter (SOM) is an important component of ecosystem carbon stocks. Generally, SOM found in mineral and organo-mineral soils can be categorised into two fractions: particulate organic matter (POM) and mineral-associated-organic matter (MAOM), both of which contain soil organic carbon (SOC). Understanding the relationship between SOC and SOM fractions provides insight into SOM decomposition and SOC storage potential. Here we show an intriguingly tight relationship between the fraction of SOC in SOM (denoted as f OC $$ {f}_{\mathrm{OC}} $$ ), habitat and soil physical properties, as well as SOC stored in POM and MAOM. This opens up new ways to predict spatial variations in the distribution of POC and MAOC using more widely available f OC $$ {f}_{\mathrm{OC}} $$ data as a covariate. By compiling 14 datasets and 9503 measurements from across Europe and globally we analysed f OC $$ {f}_{\mathrm{OC}} $$ across mineral and organic soils, which fell between 0.38 and 0.58, consistent with variation in carbon of major plant components. f OC $$ {f}_{\mathrm{OC}} $$ followed a habitat gradient with lowest median values in Seagrass sediments (0.36 ± 0.09) and Permafrost habitats, followed by croplands (0.47 ± 0.08) and a maximum in semi-natural habitats (e.g., neutral, acid and calcareous grasslands) (0.56 ± 0.07), with differences between broadleaved (0.50 ± 0.087) and coniferous woodlands (0.53 ± 0.07) which were driven by overall organic matter content. The data show a tight link between vegetation carbon and the contents of SOC and SOM across various habitats, which could be used to inform agricultural soil management, improved land-use planning (e.g., woodlands), and tracking climate-related SOC targets.

Dehesas are mosaics of open grassland and standalone trees that are diversity reservoirs. However, they have recently faced abandonment and intensification, being replaced by plantations of fast-growing trees or subject to encroachment. Following a change in dehesa communities and structure, a change in soil microbial diversity and functionality in dehesas is expected, but dehesas’ microbial diversity is still a big unknown. In this work, we bring to light the soil prokaryotic taxonomic diversity in dehesa ecosystems and present a first approach to assessing their metabolic diversity through metabarcoding data. For this, we compared three dehesas dominated by different tree species: (i) one dehesa dominated by Quercus ilex; (ii) one dominated by Pinus pinea; and (iii) one dominated by a mixture of Q. ilex and Q. suber. At each dehesa, samples were taken under the canopy and in the open grassland, as well as through two seasons of peak vegetation productivity (autumn and spring). Our results show the following findings: (1) seasonality plays an important role in prokaryotic richness, showing higher values in autumn, and higher evenness in spring; (2) the effect of seasonality on the soil’s prokaryotic diversity is often modulated by the effect of tree species and canopy; (3) taxonomic diversity is driven mainly by the site effects, i.e., the opposite of the metabolic diversity that seemed to be driven by complex interactions among seasons, tree species, and canopies.

As a consequence of climate change and unsustainable management practices, agricultural soils in the Mediterranean region are often degraded. The application of composted organic wastes is traditionally considered a beneficial practice to improve soil health. We studied the permanence of the beneficial effects of the long-term (20 years) application of composted organic wastes, versus mineral fertilization or no fertilization, on Mediterranean vineyard soil health. To this aim, four years after ceasing fertilization, a comprehensive analysis of soil physicochemical and biological properties, including prokaryotic diversity and functional traits by genome-centric metagenomics, was conducted to ascertain whether the beneficial effects of organic fertilization were maintained 4 years after its end. In general, but not always, soils treated with composted organic wastes showed significantly higher values of many physicochemical and biological properties (e.g., organic matter, Olsen phosphorus and extractable potassium), resulting in improved soil multifunctionality. However, despite such statistical significance, the quantitative magnitude of many of those differences was small. Fertilization regimes had a lasting strong influence on soil prokaryotic communities, as 70% of the 200 metagenome-assembled genomes and 86% of the prokaryotic functional traits showed significant differences among treatments. Organic amendments promoted nitrifying taxa such as Nitrosocosmicus oleophilus and Nitrospira japonica . In minerally-fertilized soils, resource acquisition and stress tolerance strategies were fostered among prokaryotic communities, possibly due to resource limitation and soil degradation, respectively. Stress tolerance traits were lower under organic fertilization, likely due to the improved soil properties and functions. The results obtained suggest that soil functions are influenced by microbial genes involved in nitrogen and carbon cycling, underscoring the central role of microbial metabolism in sustaining soil health and ecosystem functioning. This study demonstrates the lasting benefits of composted organic amendments in promoting soil multifunctionality in vineyard soils. ### Competing Interest Statement The authors have declared no competing interest. Ikermugikortasuna mobility programme of the Basque Government MICIU/AEI/10.13039/501100011033, ReCROP PCI2021-121935, VinAE PCI2025-163141 European Union, https://ror.org/019w4f821, No. 101086179 (AI4SoilHealth)

Soil contamination by antibiotics is a global issue of great concern that contributes to the rise of bacterial antibiotic resistance and can have toxic effects on non-target organisms. This study evaluated the variations of molecular, cellular, and histological parameters in Eisenia fetida earthworms exposed to sulfamethazine (SMZ) and tetracycline (TC), two antibiotics commonly found in agricultural soils. The earthworms were exposed for 14 days to a series of concentrations (0, 10, 100, and 1000 mg/kg) of both antibiotics. SMZ and TC did not affect the survival of E. fetida, , however, other effects at different levels of biological complexity were detected. The two highest concentrations of SMZ reduced the viability of coelomocytes. At the highest TC concentration, there was a noticeable decline in cell viability, acetylcholinesterase activity (neurotoxicity), and the relative presence of mucopolysaccharides in the epidermis (mucous production). Glutathione S-transferase activity decreased in all TC treatments and at the highest SMZ concentration. However, levels of malondialdehyde and protein carbonyls did not change, suggesting an absence of oxidative stress. Tetracycline was neurotoxic to E. fetida and changed the integrity of the epidermis. Both antibiotics altered the intestinal microbiota of E. fetida, , leading to a reduction in the relative abundance of bacteria from the phyla Proteobacteria and Bacteroidetes, while causing an increase in the phylum Actinobacteroidota. All observed changes indicate that both SMZ and TC can disrupt the earthworms' immune system and gut microbiome, while fostering the growth of bacteria that harbour antibiotic resistance genes. Finally, both antibiotics exerted additional metabolic and physiological effects that increased the vulnerability of E. fetida to pathogens.

The remediation of mixed contaminated soil is challenging as it often requires actions to minimize metal-induced risks while degrading organic contaminants. Here, the effectiveness of different bioremediation strategies, namely, rhizoremediation with native plant species, mycoremediation with Pleurotus ostreatus spent mushroom substrate, and biostimulation with organic by-products (i.e., composted sewage sludge and spent mushroom substrate), for the recovery of a mixed contaminated soil from an abandoned gravel pit was studied. The combination of biostimulation and rhizoremediation led to the most significant increase in soil health, according to microbial indicator values. The application of composted sewage sludge led to the highest reduction in anthracene and polychlorinated biphenyls concentrations. None of the strategies managed to decrease contamination levels below regulatory limits, but they did enhance soil health. It was concluded that the biological remediation treatments improved soil functioning in a short time, before the concentration of soil contaminants was significantly reduced.

Landfills and waste disposal sites in the Basque Country are summarized in the inventory of soils that either currently support or have supported potentially polluting activities or facilities (Law 4/2015). Notably, “Landfill 17,” located in Gernika-Lumo, has been receiving, for decades, sewage sludges from the local wastewater treatment plant (WWTP) as agricultural amendment. In order to decontaminate and recover soil functionality, a combination of bioremediation (which involved bioagumentation and phyto- and vermitechnologies) and complementary bioremediation strategy (i.e., promotion and maintenance of the native vegetation) was implemented in situ. Physicochemical and ecotoxicological characterization were achieved. Furthermore, an ecotoxicological assessment of the soils upon flora and fauna was carried out through the application of different bioassays and biomarkers. Additionally, an integrative biomarker response (IBR/n) index was calculated to provide a holistic view of the soil general status. Critical pollutants [Cd, Cr, Ni, Pb, benzo(a)pyrene, and dieldrin] were observed in most of the treated sites. Microbial parameters did not present remarkable differences among sites. However, plant indicators pointed the non-treated site (MN8) as the unhealthiest. This was also observed in earthworms’ immune system, where cytotoxicity appears when exposed to non-treated soils. In conclusion, this field study showed that the combination of bioaugmentation, phytoremediation with native species, and vermiremediation is highly useful in eliminating mixed contamination, improving soil health, and ultimately restoring ecosystem functionality and biodiversity.

1. In anthropic savanna ecosystems from the Iberian Peninsula (i.e. dehesa), complex interactions between climate change, pathogen outbreaks and human land use are presumed to be behind the observed increase in holm oak decline. These environmental disturbances alter the plant-soil microbial continuum, which can destabilize the ecological balance that sustains tree health. Yet, little is known about the underlying mechanisms, particularly the directions and nature of the causal-effect relationships between plants and soil microbial communities.2. In this study, we aimed to determine the role of plant-soil feedbacks in climate induced holm oak decline in the Iberian dehesa. Using a gradient of holm oak health, we reconstructed key soil biogeochemical cycles mediated by soil microbial communities. We used quantitative microbial element cycling (QMEC), a functional gene-array-based high-throughput technique to assess microbial functional potential in carbon, nitrogen, phosphorus and sulphur cycling.3. The onset of holm oak decline was positively related to the increase in relative abundance of soil microbial functional genes associated with denitrification and phosphorus mineralization (i.e. nirS3, ppx and pqqC; parameter value: 0.21, 0.23 and 0.4; p < 0.05). Structural equation model ( chi(2)= 32.26, p-value = 0.73), more over, showed a negative association between these functional genes and soil nutrient availability (i.e. mainly mineral nitrogen and phosphate). Particularly, the holm oak crown health was mainly determined by the abundance of phosphate (parameter value = 0.27; p-value < 0.05) and organic phosphorus (parameter value = -0.37; p-value < 0.5).4. Hence, we propose a potential tree-soil feedback loop, in which the decline of holm oak promotes changes in the soil environment that triggers changes in key microbial-mediated metabolic pathways related to the net loss of soil nitrogen and phosphorus mineral forms. The shortage of essential nutrients, in turn, affects the ability of the trees to withstand the environmental stressors to which they are exposed.

Anthropogenic activities have resulted in the buildup of contaminants in the soil, posing risks to ecosystems and human health. The revitalization of contaminated sites from former industrial activities is a matter of much interest and concern since they are usually located in urban or peri-urban areas. The transformation of brownfields into greenfields normally requires remediation interventions that address soil contamination. Traditional physicochemical techniques of soil remediation often involve a high economic cost and environmental disturbance. Operational costs and more stringent environmental regulations at disposal sites are making on-site and, above all, in-situ biological remediation technologies more attractive. The aim of biological remediation techniques is not only to decrease the concentration of soil contaminants, but also to recover soil health and the provision of ecosystem services. The selection of the most suitable biological remediation strategies depends on a variety of factors, such as the nature of the contaminants, their concentration and bioavailability, the depth of contamination, soil properties and edaphoclimatic conditions, economic cost, environmental policies, and so on. Among the different biological remediation strategies, this chapter provides a review of plant-based remediation strategies (phytoremediation, phytomanagement) for the remediation of contaminated soils, including microbial-assisted phytoremediation. In addition, we present a case study on the application of compost-assisted phytoremediation, using intercropping with poplar trees and alfalfa, for the recovery of an industrial contaminated soil.

Abstract The biological remediation of mixed contaminated soils is exceedingly challenging as it often requires actions to minimize metal-induced risks while degrading organic contaminants. In this study, we assessed the effectiveness of three biological remediation options (rhizoremediation, biostimulation, mycoremediation), applied together or separately, for the recovery of a mixed contaminated soil from a gravel pit. We evaluated the effectiveness of these remediation strategies in terms of both reduction in soil contamination levels and recovery of soil health. The combination of biostimulation (by the application of composted sewage sludge) and rhizoremediation with Medicago sativa plants resulted in the highest recovery of soil health. The application of the composted sewage sludge led to the highest reduction in anthracene and polychlorinated biphenyls concentrations. Still, the studied biological remediation options did not manage to decrease soil contamination levels below some of the limits set by the regional legislation. By contrasts, they enhanced soil health, as reflected by the values of soil microbial indicators, pointing out to the fact that they can stimulate soil functioning in a relatively short period of time, long before the concentration of soil contaminants is significantly reduced.

Abstract The biological remediation of mixed contaminated soils is exceedingly challenging as it often requires actions to minimize metal-induced risks while degrading organic contaminants. In this study, we assessed the effectiveness of three biological remediation options (rhizoremediation, biostimulation, mycoremediation), applied together or separately, for the recovery of a mixed contaminated soil from a gravel pit. We evaluated the effectiveness of these remediation strategies in terms of both reduction in soil contamination levels and recovery of soil health. The combination of biostimulation (by the application of composted sewage sludge) and rhizoremediation with Medicago sativa plants resulted in the highest recovery of soil health. The application of the composted sewage sludge led to the highest reduction in anthracene and polychlorinated biphenyls concentrations. Still, the studied biological remediation options did not manage to decrease soil contamination levels below some of the limits set by the regional legislation. By contrasts, they enhanced soil health, as reflected by the values of soil microbial indicators, pointing out to the fact that they can stimulate soil functioning in a relatively short period of time, long before the concentration of soil contaminants is significantly reduced.

The use of animal manure as organic fertilizer is a common agricultural practice that can improve soil health and crop yield. However, antibiotics and their metabolites are often present in animal manure and, hence, in manure-amended soil. The aim of this study was to assess the induced development of oxytetracycline (OTC) tolerance in soil bacterial communities as a result of the addition of OTC to soil amended with well-aged cow manure. To this purpose, soil amended with well-aged cow manure was repeatedly – three times – spiked with different OTC concentrations (0, 2, 20, 60, 150, and 500 mg OTC kg−1 dry weight soil, each time) according to a pollution-induced community tolerance (PICT) assay. The PICT detection phase was conducted in Biolog EcoPlatesTM in the presence of the following OTC concentration gradient in the wells: 0, 5, 20, 40, 60, and 100 mg L−1. For all treatments, the application of OTC in the PICT selection phase resulted in lower values of bacterial metabolic activity (i.e., lower values of average well color development) in the PICT detection phase. A significant increase in OTC tolerance was observed in soil bacterial communities that had been exposed three times to ≥ 20 mg OTC kg−1 DW soil during the PICT selection phase. In general, higher levels of OTC exposure during the PICT selection phase resulted in bacterial tolerance to higher OTC concentrations during the PICT detection phase, pointing to a dose-dependent induced tolerance. It is important to (i) rationalize the amount of antibiotics administered to livestock, and (ii) treat properly the antibiotic-containing manure before its application to agricultural soil as fertilizer.