Plant and Soil

Soil microbial communities have a variety of mechanisms to deal with emerging drought stress. One well-documented mechanism is increased microbial production of extracellular polymeric substances (EPS), which can potentially change the soil density and water holding capacity. Yet little is known about how microbial diversity influences the functional capacity of EPS formation and the resulting outcomes in water dynamics. To understand more about communal microbiome EPS production, we set up sterile mesocosms where we examined the effects of microbial diversity (high or low treatments) and nutrient input (supplement or deficient treatments) on these processes. To capture the microhydrology of the mesocosms, we measured water holding (WH), infiltration, evaporation, and soil properties we believe microbes are altering (EPS, soil aggregation). Our hypothesis stated that if diversity was artificially manipulated, then soil-water properties will be altered via production of EPS. We predicted that low diversity systems would have lower functional diversity, leading to less EPS production, moisture storage, and minimal changes from inert soil media. As predicted, we found that the high-diversity systems had a higher water retention and lower rates of water loss over time than low-diversity systems. This trend was magnified in the nutrient-supplemented treatment, suggesting that EPS production and subsequent water-holding traits are emergent features of the microbiome. Unexpectedly, we observed a correlation between the amount of water retained and the quantity of lipid EPS produced. This suggests that EPS composition, rather than quantity, is determinative of a biofilms function. In conclusion, it appears that microbial diversity influences soil properties that are important to moisture retention within these systems. To date, the role that microbes and their diversity play in soil hydrology has been severely understudied, so this work aims to build ecological understandings of these systems. These findings are valuable, for if we learn how microbes manipulate soil moisture, we can apply these functions to advance sustainable agricultural practices and enhance ecosystem resilience to water scarcity in arid regions. Open Research StatementUpon publication data, and code will be made available through Zenodo. Sequencing data will be uploaded to NCBI SRA.

O_LIA productivity-driven higher nutrient demand of trees in diverse mixtures is frequently reported. Yet, it remains unclear how tree diversity influences microorganisms-plants interactions, in which microbes facilitate tree nutrient acquisition in exchange for carbon (C) to meet the resource demand of both. C_LIO_LIUsing a long-term tree diversity experiment in the subtropics, we assessed microbial investment in C-, nitrogen (N)-, and phosphorus (P)-acquiring enzymes in litter and mineral soil, testing the effects of tree species richness and mycorrhizal type (arbuscular (AM)- vs. ectomycorrhizal (EcM)-associated tree species). C_LIO_LIWith increasing tree species richness, microbial investment in C acquisition decreased, while investment in N and/or P acquisition increased in litter and in mineral soil. In mineral soil of AM-associated tree mixtures, ecoenzymatic stoichiometry revealed a shift from microbial investment in C toward P acquisition as tree species richness increased. C_LIO_LIOur findings suggest that tree diversity strengthens microbe-tree interactions in terms of C-for-nutrient exchange. This highlights the key role of soil microorganisms, particularly in AM symbiosis, shaping tree diversity-biogeochemical feedbacks. C_LI

Plant roots are broadly colonized by endophytic fungi with saprotrophic capabilities, but our understanding of whether they function in ways that are beneficial or detrimental to the host remains limited to model organisms. We hypothesized that endophytic fungi broadly affect plant access to soil nutrients, particularly organic forms that are typically not directly available to the plant. To address this, we paired 41 fungal endophytes with switchgrass (Panicum virgatum L.) and provided either inorganic or organic forms of nitrogen (N) and phosphorus (P). We evaluated how the fungi affected plant tissue N and P as well as plant growth. We also examined if these outcomes could be predicted from fungal phylogenetic relationships, in vitro traits of the fungi, or characteristics of the habitat from which fungi were isolated. There was substantial variation in both plant N (0.05-0.63%) and P (0.02-0.10%) acquisition that depended on the interaction of fungus and nutrient treatment. More fungi were beneficial for plant N than for P and shoot nutrients generally increased more than root nutrients from fungal associations. However, fungal effects on plant nutrients were not predicted by fungal traits, habitat traits, or fungal phylogenetic relationships. This unpredictability highlights a key challenge for incorporating endophytes into nutrient management strategies. Improving our ability to predict endophyte impacts on host nutrient acquisition will require identifying the mechanisms underlying observed beneficial effects and scaling up to realistic, diverse root microbial communities.

The xerophyte argan (Sideroxylon spinosum) has great ecological and socioeconomic importance for Morocco. However, it is endangered due to climate change and human overexploitation. We assess drought resistance traits of argan and test the potential of arbuscular mycorrhizal (AM) symbiosis to promote its growth and mitigate the consequences of drought. We compare ten endemic Moroccan mycorrhizal inocula with the model AM fungus Rhizophagus irregularis and with the drought-adapted isolate Diversispora omaniana. We integrated physiological phenotyping and RNA sequencing to investigate the stress resistance mechanisms of argan against drought. We show that AM symbiosis significantly mitigates drought effects on plant growth, mainly by improving water relations and photosynthetic efficiency, resulting in increased growth rates. Taken together, physiological and transcriptomic analyses show that stress markers were moderatly induced during severe drought stress irrespective of mycorrhizal status, suggesting that argan adopts a drought-coping strategy that involves both, stress avoidance and stress tolerance. Argan is highly AM-responsive, both at the phenotypic and transcriptomic level, suggesting that AM has great potential to promote argan growth under drought stress.

To investigate the differences in soil nutrients and microbial community structure in the rhizosphere between healthy and diseased Zingiber officinale plants, soil samples were collected from healthy root-zone soil (ZSH), healthy rhizosphere soil (RSH), diseased root-zone soil (ZSD), and diseased rhizosphere soil (RSD). Diseased soils had significantly higher pH values, whereas RSH showed the strongest acidity. Moreover, pH, AN, and AK contents in diseased soils were significantly higher than those in healthy soils, while SOM and AP were significantly lower. The -diversity of microbial communities in diseased soils was significantly reduced, and the community structure was distinctly differentiated from that of healthy soils. In diseased soils, the abundance of potential pathogenic taxa such as Ralstonia solanacearum increased significantly, while beneficial genera such as Bradyrhizobium decreased. Redundancy analysis and correlation analysis indicated that soil pH, AN, SOM, and AP were the major environmental factors driving changes in microbial community structure. The occurrence of soil-borne diseases in Zingiber officinale is closely associated with soil nutrient imbalance and disruption of microbial community structure. The study identified candidate microbial taxa (e.g., beneficial Sphingomonas, Streptomyces) and key soil properties (pH, available nitrogen) that differentiate healthy from diseased ginger soils. Together, these findings provide a theoretical basis for improving diseased soils through microecological regulation strategies, and also serve as a foundation for generating testable hypotheses in future hypothesis-driven research on ginger soil-borne disease suppression.

Crop species may shape soil chemical properties and root-associated microbiota, but direct comparisons between contrasting crops remain limited. We compared soils and root-associated bacterial communities of Welsh onion (Allium fistulosum) and sweet potato (Ipomoea batatas) under the same field context. Sweet potato soil showed significantly lower electrical conductivity, inorganic nitrogen, and Mg saturation than control soil. Root-associated communities differed between crops, whereas alpha diversity did not. Proteobacteria-related taxa were more represented in Welsh onion roots, whereas Actinomycetia-related taxa were more represented in sweet potato roots, providing a basis for future studies on crop-specific soil microbial management.

Commercial inoculants based on associative diazotrophs and methylotrophic bacteria, and bios-timulant programs, are marketed for broad use in cereal and fiber production, yet most field deployment occurs over already adequate mineral fertilization, where their marginal value is poorly quantified. We reanalyzed three randomized complete-block trials conducted at commercial scale in the Brazilian Cerrado (Sao Desiderio, Bahia) under full conventional fertilization: corn (seven treatments, four blocks; 2023/24), cotton (five treatments, four blocks; 2024/25), and soybean (four treatments, six blocks; 2024/25). Treatments evaluated Azospirillum brasilense, Methylobacterium symbioticum, Bradyrhizobium spp., a Bacillus phosphorus solubilizer, and biostimulants, applied via seed and foliar routes. Beyond conventional analysis of variance, we fit mixed models with block as a random effect, quantified effect sizes and coefficients of variation, computed the minimum detectable difference at 80% power, and applied two one-sided equivalence tests (TOST) against the untreated control at margins of plus or minus 10% and 15% of control yield. No treatment produced a statistically significant yield gain in any crop (all p greater than 0.8; fixed and mixed models concordant). In cotton, the best-powered trial (coefficient of variation 6%), all treatments were statistically equivalent to the untreated control within a 10% margin, an affirmative negative result. In corn and soybean the trials were underpowered (minimum detectable difference 21 to 26% of control), so non-significance is not equivalence; corn showed non-significant numerical gains up to 8.9% with phosphorus-solubilizer and methylotroph combinations that could not be excluded. Foliar nutrient concentrations (single composite per treatment, descriptive) showed no enrichment in inoculated treatments. Under standard fertilization, these commercial inputs delivered no detectable agronomic value where the data were adequately powered to test it. The results clarify the low empirical bar that current associative-fixation products meet in the field and, by extension, the agronomic threshold that engineered, plant-controlled nitrogen fixation must exceed to be useful.

Plants form holobionts by associating with diverse microbiota. Self-organization gives rise to emergent properties of the holobiont, such as increased resistance to pathogens. However, the local factors contributing to the self-organization are not well understood. We hypothesized that nematode communities and their associated microbiota govern the rhizobiome of the model plant holobiont tomato in terms of its suppression of root invasion by the parasite Meloidogyne hapla, and that the soil legacy influences the suppressive potential mediated by these biota. In pot experiments, a resistant tomato holobiont was favored by assembly in the presence of a nematode community conditioned by tomato plants, compared to oilseed rape or fallow soil. Nematode communities conditioned by tagetes could enhance resistance even better than tomato. Microbiota from crushed tomato-conditioned nematode communities increased resistance of the tomato holobiont, compared to microbiota of nematode communities conditioned by maize, or heat-inactivated microbiota. The 0.2 micrometre filtered microbiota from crushed nematodes had the same effect, suggesting a role of nematode-associated bacteriophages in holobiont assembly. The results indicate that soil nematodes and their associated microbiota play a role in the local organization and stabilization of plant holobionts. They can influence the resistance of plants that subsequently grow in the same soil. From an applied perspective, crop rotation schemes that alter nematode-microbiota communities could be harnessed to engineer crop holobionts.

O_LIPlants take up nutrients from the soil while investing in absorptive root surface or mycorrhizal partners. Root hairs - a major structure for nutrient uptake and cheap to build - increase the absorptive root surface. As such they are an important component of plant resource economics but largely neglected in root economic concepts so far. C_LIO_LIThis is mainly due to data scarcity, which we set out to overcome by measuring root-hair traits on 82 European grassland species in a greenhouse experiment. Using fluorescence and light microscopy, root-hair length and incidence was measured along with mycorrhizal colonization. C_LIO_LIWe found a phylogenetically conserved trade-off between plant investment into root hairs and mycorrhiza. A similar trade-off between root-hair incidence and mycorrhiza occurred at the intraspecific level, while patterns were heterogeneous among species. Plant species with high colonization rates showed the highest variability in root-hair incidence. C_LIO_LIWe conclude that plants vary along a gradient ranging from investment into root hairs as part of a "do-it-yourself" strategy to collaboration with mycorrhizal fungi while showing intraspecific variation in root-hair incidence. These findings demonstrate that root hairs play a fundamental role in fine-root trait variation and need to be considered when studying belowground plant economic strategies. C_LI

The Digital Pedon (DP) is an open-source Python framework that represents a soil profile as a continuously updated digital twin, bridging three persistent gaps in soil science: disconnected models and observations, cross-database interoperability, and the inference gap between raw sensor signals and agronomically meaningful variables. Integrating real-time sensor streams, model-based solver chains (Model-Zoo), GLOSIS-compliant ontology mapping, and a novel LLM agentic interface layer enabling natural language soil queries, the DP supports applications spanning precision agriculture, digital soil mapping, and environmental sustainability assessment. Four proof-of-concept experiments confirm automatic profile initialisation fidelity, solver chain consistency, ontology compliance, and user-defined solver extensibility.

Creeping bentgrass (Agrostis stolonifera) is a turfgrass species established on golf course surfaces but is criticized for high irrigation requirements. While genetic variation for water deficit stress tolerance exists between cultivars, the lack of defined critical soil water potential thresholds (Soil {Psi}crit) for this species complicates precise irrigation strategies and benchmarks for plant breeding. This study utilized a polyethylene glycol (PEG) infused agar-based system to simulate water potential reductions and determine the water potential threshold ({Psi}crit) for seedling root elongation. Creeping bentgrass cv Pure distinction seedlings were subjected to six water potentials ({Psi}) ranging from -0.36 MPa (no PEG applied) to -1.72 MPa. Daily digital imaging was used to measure root elongation over 5 days. Results across two experiments demonstrated that creeping bentgrass seedlings are highly sensitive to mild reductions in {Psi}. A reduction to -0.61 MPa significantly decreased root length and growth rates by over 50% compared to the control. Regression models predicted that a {Psi}crit of approximately -0.45 MPa reduced daily root growth by 25%, while upwards {Psi} of -1.0 MPa resulted in a 75% reduction of seedlings root growth. Furthermore, seedlings exposed to the lowest water potentials were predicted to require an additional 30 to 46 days to achieve the same root length as control plants. These findings establish specific {Psi}crit benchmarks for water deficit stress tolerance using a PEG-based system to induce dehydration. These methods can be used in breeding programs, and will help develop more accurate experiments examining the mechanisms of water deficit stress tolerance.

Early management decisions in operational forestry are critical for plantation success because it strongly influences seedling quality at planting. Beyond shaping seedling morphology, nursery inputs can also restructure root-associated fungal communities which has consequences for nutrient acquisition, stress tolerance and disease suppression after planting. In this study, we altered nutrient and fungicide inputs based on mycorrhizal ecological theory and quantified the effects of these treatments on key dimensions of the growth performance of radiata pine seedlings. In parallel, we profiled the root-associated mycobiome, assigning fungal taxa to functional guilds and summarizing their richness, diversity, relative abundance and community structure. Using a composite performance index that integrates the key morphological and health measures into a single response variable, together with conventional statistical models with machine learning approaches, we identified management practises that promote both plant performance and a favourable root fungal community and determined the consistent microbiome changes linked to overall quality of the seedlings. These results suggest that microbial feedback loops occur even in highly managed nursery conditions. More broadly, by combining a composite performance index with predictive modelling, we provide a practical way to test complex management combinations and identify microbiome features associated with high-quality planting stock.

Root-associated microorganisms play a pivotal role in helping plants adapt to drought stress. However, the underlying mechanisms of the rhizospheric microbiome under limiting soil moisture remain largely unresolved. Integrating total and active microbiome analyses enables a more accurate interpretation of microbial responses to climate change-associated water stress. We assessed the effect of reduced rainfall on two wheat genotypes, drought-tolerant (DT) and drought-sensitive (DS), using rainout shelters that allowed 100%, 75%, 50%, and 25% of natural precipitation to reach the crop. At the peak of the growing season, rhizosphere samples were collected for metagenomic (MG) and metatranscriptome (MT) sequencing. In parallel, rhizosphere volatile organic compounds (VOCs) were collected and analysed. Differential expression analysis of metatranscriptomic data using metagenomic abundance as a cofactor was performed by comparing all treatments to the 100% precipitation control. Our results demonstrate that particularly oxidative stress-related transcripts intensify in DS as rainfall decreases. Transcriptomic shifts primarily involved upregulation of transcripts associated with antioxidant (catalase, superoxide dismutase), heat shock proteins (Hsp10, Hsp60, DnaK/DnaJ, GroEL, GroES), as well as microbial functions related to osmoregulation, proline and glycine betaine (PutA, PutP, OpuBB), and plant growth-promoting traits such as auxin production, phosphate solubilization. Moreover, volatile organic compound (VOC) emissions differed significantly between the control and drought treatments, with higher emissions, particularly acetates, in the DS genotype than in the DT genotype. Overall, pronounced drought-induced shifts in active microbial functions and VOC emissions indicate high sensitivity and functional plasticity of the active microbiome, whereas the total microbiome remains robust under medium drought.

Drought threatens food security globally. Adaptive root phenotypes and microbiomes can improve maize (Zea mays L.) water uptake and tolerance to drought. However, synergisms between root phenotypes and microbiomes remain underexplored. We aimed to investigate the association between varying root phenotypes and rhizosphere microbiomes under field-scale drought. We grew 22 maize inbred lines in the field under optimal water availability and drought imposed by excluding rain with rainout shelters. We quantified grain yield and measured root architectural and anatomical phenotypes on root crown and cross-section images obtained by laser ablation tomography, respectively. We characterized rhizosphere prokaryotic and fungal communities with DNA metabarcoding of ribosomal markers. Rhizosphere microbial diversity predominantly associated with root anatomy rather than root architecture. Cortical parenchyma wall width explained 13.1% of the variance of the prokaryotic {beta}-diversity and correlated with grain yield under control conditions. Under the same conditions, number of cortical cell files and metaxylem vessels explained 1.4-2.1% of the variance of prokaryotic and fungal {beta}-diversities. No effect of the root phenotypes was observed under drought. We found 248 significant correlations between microbial taxa abundances and root anatomical phenotypes, especially cortex-related phenotypes such as number of cell files and living cortical area. Overall, a greater number of correlations was found under control conditions. We identified root phenotypes explaining a small but significant percentage of the variance of the microbial {beta}-diversity, mostly under optimal water availability. We showed that especially root anatomy is associated with rhizosphere microbial diversity in field-grown maize.

Rhizosphere oxidation is a key adaptive mechanism in reductive soil environments, in which oxygen released from roots alters rhizosphere redox conditions and regulates biogeochemical processes. Rice plants possess an internal oxygen transport system, and radial oxygen loss (ROL) from roots is closely associated with root development. However, the spatial patterns of ROL in soil and their relationships with root traits remain poorly characterized. In this study, we developed a multimodal imaging system that integrates planar oxygen optodes with X-ray computed tomography to simultaneously visualize rhizosphere oxidation and root development in rice. Daily time-course tracking of individual crown roots revealed dynamic changes in the spatial distribution and magnitude of rhizosphere oxygen in relation to root elongation and aging. Root thickness was positively correlated with dissolved oxygen levels near root tips. Genotypic comparisons further identified a cultivar with reduced rhizosphere oxidation despite possessing thicker roots among the tested genotypes, thereby indicating the involvement of additional physiological processes. Overall, these findings demonstrate that rhizosphere oxidation is regulated by root growth stage and thickness and dynamically modulated during root development.

The composition and quantity of root exudates are strongly influenced by physiological and environmental conditions, reflecting dynamic changes in plant metabolism. Although many studies report that root exudate metabolite profiles vary with plant phenology, few have disentangled the effects of phenological stage from those of nutrient availability. We collected root exudates from white lupin (Lupinus albus) grown in a fine phosphorus (P) gradient (5, 10, 20, 30 and 50 {micro}M P) in hydroponics at three developmental stages, performed untargeted metabolomics using GC-MS, and measured 10 above and belowground traits. Our results show that plant phenological stage exerts a stronger influence on exudate metabolomic profiles than variation in P supply. During leaf development, exudates were dominated by metabolites associated with carbon metabolism, whereas flowering was characterized by compounds related to secondary metabolism and cell wall turnover. Phosphorus influenced exudate profiles only at the flowering stage, with distinct profiles observed at 5 and 10 {micro}M P compared with 20-50 {micro}M P. These findings provide new insights into the temporal regulation of root exudation and demonstrate that plant developmental stage is a primary determinant of metabolic responses to phosphorus availability and, potentially, rhizosphere functioning under nutrient limitation. HighlightRoot exudate quantity and composition in white lupin is shaped more by plant phenological stage than phosphorus supply, with phosphorus effects emerging only during flowering.

Plant-associated bacteria can improve plant performance under abiotic stress, but beneficial functions in plant microbiomes may depend on defined combinations of microorganisms rather than individual isolates alone. Here, we developed a cube-based screening strategy to identify functional synthetic microbial communities (SynComs) from 135 quinoa-associated bacterial isolates while preserving combinatorial diversity and traceability of isolate-level contributions. The isolates were divided into five 27-isolate sets, each arranged as a 3 x 3 x 3 cube in which each 3 x 3 layer was defined as a 9-isolate SynCom, generating 45 SynComs in total. Screening under 100 mM NaCl identified SynCom DY1 (SCDY1) as a candidate salt stress-mitigating consortium. SCDY1 consisted of nine taxonomically diverse isolates and exhibited a multifunctional profile, including siderophore production, phosphate solubilization, carboxymethyl cellulose degradation, indole compound production, and growth under saline conditions. In Arabidopsis thaliana, SCDY1 promoted primary root elongation and biomass accumulation in a salinity-dependent manner, with the clearest effect under 120 mM NaCl, and at least a subset of constituent bacteria was recoverable from inoculated seedlings. RNA sequencing and targeted RT-qPCR indicated that SCDY1 modulated host gene expression under moderate salinity stress, with responsive genes associated with oxidative stress, water- and oxygen-related processes, phenylpropanoid biosynthesis, glutathione metabolism, and root epidermis-related processes. Root hair phenotyping further showed that SCDY1 enhanced root hair-related traits and shifted visible root hair formation closer to the root apex. These findings identify a quinoa-derived SynCom that improves plant performance under salinity stress and provide a practical, traceable framework for discovering beneficial microbial consortia from plant-associated bacterial collections. Scope statementThis manuscript fits the Research Topic "Harnessing Plant Microbiomes for Climate Resilience: From Ecological Insight to Synthetic Community Design" in Frontiers in Plant Science because it presents a traceable strategy for discovering functional synthetic microbial communities from a stress-adapted plant-associated bacterial collection. We developed a cube-based screening strategy using 135 quinoa-associated bacterial isolates and identified a nine-isolate synthetic microbial community, SCDY1, that promotes Arabidopsis growth under moderate salinity stress. The study integrates microbiological screening, characterization of plant growth-promoting traits, bacterial re-isolation, plant growth phenotyping, RNA-seq, RT-qPCR, and root hair phenotyping. These analyses link SCDY1 treatment to salinity-dependent growth promotion, recoverable bacterial members, stress- and redox-associated transcriptional changes, phenylpropanoid-related responses, and modulation of root epidermal phenotypes. By connecting a defined SynCom with host transcriptional and root epidermal responses, this work advances understanding of beneficial plant-microbe interactions under salt stress. The cube-based design also provides a practical and traceable framework for discovering functional SynComs from large plant-associated bacterial collections, which should be of interest to researchers studying plant symbiosis, microbiome engineering, abiotic stress tolerance, and sustainable crop improvement.

Beneficial interactions between plants and microorganisms strongly influence plant health and productivity, and root exudates play a central role in shaping these associations. This study analyzed the transcriptional responses of the bacterial endophytes Enterobacter asburiae RCA24 and Kosakonia sacchari RCA25 to root exudates from two commercial Italian rice accessions (Oryza sativa Baldo and Vialone Nano) and from an accession of the wild progenitor of tropical rice, Oryza rufipogon. Bacterial transcriptome analyses revealed that RCA24 responds differently to O. sativa varieties and that RCA25 was more stimulated by O. rufipogon. Changes in bacterial gene expression were mainly related to central metabolism, stress response, and signal transduction, highlighting a precise pattern of interaction. On the other hand, transcriptome analysis of inoculated rice revealed that RCA24 triggered broader transcriptional changes in plants than RCA25. Differentially expressed genes were related, especially in shoots, to defense responses, hormone-mediated signaling, and ribosome biogenesis, revealing that plants discriminate bacterial strains in a genotype-specific manner at the transcriptional level. Our findings suggest that traits beneficial to plant-soil microbiota interactions present in O. rufipogon and lost during domestication and diversification could be identified and reintroduced into modern rice varieties to improve sustainable field performance through beneficial microbial associations.

Modern agriculture is characterized by substantial fertilizer nitrogen (N) losses from soils, resulting in low crop N-use efficiency. Biological nitrification inhibitors (BNIs) are studied as a strategy to improve N retention in soils by suppressing nitrification. However, the impacts of applying exogenous BNIs to crops with unknown intrinsic BNI capacity remain poorly understood. In this study, we evaluated the impacts of adding three BNIs (methyl 3-(4-hydroxyphenyl) acrylate [MHPA], 6-methoxy-2(3H)-benzoxazolone [MBOA], and limonene), their mixture, and the synthetic nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on barley (Hordeum vulgare L.) growth, plant and soil N dynamics, and soil microbial communities. Using a rhizobox system with planted and bare-soil compartments, combined with 15N isotope tracing and molecular microbial community analyses, we assessed the spatio-temporal dynamics of N transformations, losses, plant N uptake, and microbial community responses in an alkaline agricultural soil. Independent of inhibitor application, the applied fertilizer N was lost primarily through NO- leaching (3-9% of the applied N). In contrast, N2O emissions represented only 0.001-0.028% of the applied N and varied with inhibitor type. MHPA increased dissolved inorganic N soil pools without affecting plant biomass or 15N uptake or strongly shifting microbial community composition. MBOA reduced NO3- concentrations in soil pore water without influencing plant growth or N uptake but shifted soil microbial community composition. In contrast, limonene reduced plant growth and 15N uptake and most significantly altered microbial community composition, without significantly changing N availability. Applying a BNI mixture, as well as limonene alone, was detrimental to plant growth and 15N uptake. DMPP showed only minor effects on N pools, plant growth, plant N uptake and microbial community composition. Overall, our results reveal both the potential and limitations of exogenous BNI application for improving N retention in crop systems.

Atmospheric inorganic nitrogen (N) deposition has been linked to increased tree phosphorus (P) deficiency and shifts in ectomycorrhizal (ECM) fungal community composition across Europe, but the underlying mechanisms remain poorly understood due to the scarcity of species-level studies of fungal physiology at large spatial scales. Here, we characterized ECM communities in nine Scots pine (Pinus sylvestris L.) stands across Europes largest N deposition gradient to gain mechanistic insight into N-driven ECM community shifts, by assessing morpho-physiological traits (i.e. soil exploration types and ECM root-tip level exoenzyme activities involved in organic N and P acquisition) on individual ectomycorrhizas. Our data revealed high functional variation in foraging strategies across species and sites, including within dominant ECM genera (Cortinarius, Elaphomyces, Lactarius, Piloderma, Russula). Shifts in community-level exoenzyme activities along the N deposition gradient were consistent with increasing P limitation, with a buffering effect of phosphomonoesterase activity on host nutritional status (i.e. reduced foliar N:P). These trends were mainly driven by interspecific differences in enzymatic profiles and species turnover along the gradient, rather than intraspecific variation within widespread species. Dominant low-biomass species in high N sites (e.g. E. citrinopapillatus, L. subdulcis, R. ochroleuca) were efficient P-foragers, with some displaying high oxidative activity, potentially hampering soil carbon storage under elevated N loads. These findings highlight the role of ECM species-specific traits in mediating ecosystem processes and can help understand the future of pine forests under chronic N pollution, with potential implications for applied forestry.