Applied Soil Ecology

Metabarcoding is a powerful tool for biodiversity comparisons, where standard-size DNA barcodes (>500 bases) offer better taxonomic resolution than shorter ones. Still, the choice of sequencing platforms and bioinformatics pipelines may strongly affect inferred diversity due to various technical biases. We assessed the relative performance of Illumina MiSeq i100 (2x500 paired-end), PacBio Revio and Oxford Nanopore MinION sequencing and bioinformatics pipelines, using full-length ITS amplicon sequencing datasets from a 103-species mock community and 45 composite soil samples. Despite numerous low-quality reads, PacBio yielded the lowest overall error rate and highest number of taxa. Illumina revealed the highest proportion of chimeric and index-switched reads, along with a strong bias towards shorter amplicons. MinION data analysed using PRONAME and Minovar - a bioinformatics pipeline presented here - had the largest proportion of low-quality data, and rare taxa were lost during data filtering and read polishing steps. Although Minovar enabled amplicon sequence variant (ASV) level precision for common taxa, we recommend clustering ASVs into OTUs. For PacBio, standard filtering approaches outperformed the ASV approach because they retained rare taxa. For Illumina, a stringent ASV approach or removal of rare OTUs would limit artefacts. Across all platforms, excess PCR cycles promoted chimeric and low-quality reads and lost quantitativity in biodiversity assessments. With moderate differences in effect sizes, all analytical approaches supported the conclusion that sampling design determines how we see soil biodiversity responses to land use. For biodiversity surveys based on the full-length ITS metabarcoding, we recommend using PacBio sequencing with standard, non-ASV pipelines.

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

Urban agriculture increasingly relies on compost-based substrates for sustainable production, yet we lack a clear characterization of how these systems respond to biological amendments aimed at introducing beneficial microbiota. Here we investigated how developmental stage and co-inoculation with arbuscular mycorrhizal fungi (AMF) and phosphate-solubilizing bacteria (PSB) reshape rhizosphere microbial function in Solanum lycopersicum grown in compost-based urban farm substrate. Using plant physiology assays, 16S rRNA amplicon sequencing, and metagenome-informed metaproteomics, we characterized tomato physiological responses and rhizosphere microbial activity during flowering and fruiting across control, single AMF, single PSB, and AMF and PSB co-inoculation treatments. Co-inoculation synergistically enriched beneficial taxa, improved fruit nutrient accumulation, elevated nutrient transporter and quorum sensing protein production, and drove stress-driven dormancy in competitively excluded taxa, with responses varying between developmental stages. Our findings establish metagenome-informed metaproteomics as essential for resolving stage-specific rhizosphere microbiome functional responses to tomato development and AMF and PSB co-inoculation.

The persistence of specialised survival spores produced by microbial pathogens represents a primary bottleneck in the management of plant diseases. In oomycetes, these spores (known as oospores) are largely impervious to chemical control, allowing them to persist in soil and initiate new infection cycles over many years. A prominent example is the soil-borne pathogen Phytophthora agathidicida, the causal agent of kauri dieback disease, where long-lived oospores hinder conservation efforts in native forests. The resilience of oospores is attributed to their thick wall composed of complex {beta}-glucan layers that render the oospores impermeable to most conventional biocides. Here we have investigated an enzyme-based approach for weakening the oospore cell wall. We searched enzyme databases to select {beta}-glucanases targeting a variety of linkages found in Phytophthora oospore walls. Eight of these {beta}-glucanases were successfully purified and tested for their digestive activity against intact oospores in vitro using a phenol-sulfuric acid assay. We showed that combining these enzymes was crucial to achieve significant digestion through synergies and additive effects. The optimal combination, comprising 1,3-, 1,6-, and 1,3(4)-{beta}-glucanases, was evaluated for its ability to permeabilise oospores to five biocides typically effective only on other, more sensitive lifecycle stages of the pathogen. Using a live/dead fluorescence assay, we observed that the effects of the membrane-targeting biocides were potentiated in oospores that were pre-treated with the {beta}-glucanase mixture. Our results highlight enzymatic cell wall permeabilisation as a promising strategy toward improved management of oospore persistence in kauri forest soils and against broader oomycete threats. KeypointsO_LIOur phenol-sulfuric acid assay can be used to screen for oospore-degrading enzymes. C_LIO_LISynergistic enzyme combinations are essential for effective oospore wall digestion. C_LIO_LIEnzyme pre-treatment sensitises oospores to membrane-targeting biocides. C_LI

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.

Global change drivers are reshaping agroecosystems and their sustained functions worldwide. While soil microorganisms underpin the resilience of these systems, the individual and interactive effects of multiple anthropogenic stressors on microbial community structure and function using large-scale field experiments remain poorly understood. Here, we utilize a full-factorial field experiment in a subtropical agroecosystem to investigate how land-use intensity, cattle grazing intensity, and altered precipitation regimes interact to shape soil microbiomes. Combining microbiome sequencing with network analyses and functional bioinformatics, we evaluated effects of these drivers on prokaryotic and fungal diversity, composition, predicted functional profiles, and community structure. Land-use intensity emerged as the primary driver of microbial responses, explaining 25% and 13% of the total variation in community composition for prokaryotes and fungi, respectively. Compared to intensively managed pastures, semi-natural pastures had significantly different community composition for prokaryotes and fungi and exhibited 22% higher fungal diversity. Semi-natural pastures were enriched with decomposer-associated taxa and metabolic pathways related to energy and lipid metabolism indicating enhanced microbial activity. Surprisingly, intensively managed pastures showed higher network modularity but lower network richness, suggesting a trade-off between community compartmentalization and complexity under intensive land management. Grazing and precipitation manipulations induced core microbiome changes within land-use intensities but had no impact on overall community structure and no significant interactions with land-use. Together, these findings suggest that long-term land-use legacies exert a persistent influence on soil microbial community structure, function, and organization, shaping the context within which other global change drivers operate in subtropical agroecosystems.

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.

Biochar amendments are increasingly applied to improve soil physical functioning and support carbon dioxide removal, but their effects on intrinsic soil thermal properties remain poorly characterised. We conducted the first global systematic meta-analysis of 19 independent studies, 231 control-biochar comparisons, and 529 property-specific effect sizes to test how biochar changes soil heat transfer and storage. Biochar reduced thermal conductivity by 17.6% (95% CI, -22.7 to -12.2), thermal diffusivity by 11.0% (-14.5 to -7.3), and volumetric heat capacity by 8.3% (-12.3 to -4.1). Gravimetric heat capacity showed no significant overall response (+3.3%; -7.6 to 15.4) but was supported by fewer studies. Negative responses were directionally consistent for thermal conductivity, diffusivity, and volumetric heat capacity. Moderator analyses showed that responses were most consistently associated with post-application bulk density and changes in bulk density, while application rate modulated response magnitude and soil texture constrained context dependence. Co-variation among thermal conductivity, thermal diffusivity, and volumetric heat capacity matched expected physical dependencies, indicating coordinated structural reorganisation rather than independent shifts in isolated parameters. These estimates describe intrinsic conductive and storage properties; field-scale soil temperature responses may also be modified by albedo, evaporation, vegetation, and surface energy balance. Improved integration of soil thermal measurements with moisture dynamics, structural changes, and carbon cycling is essential to accurately represent biochar effects in soil and land-surface models.

Subsistence agriculture in sub-Saharan Africa faces persistent productivity challenges due to low soil fertility, limited inputs, and increasing climate variability. Agroforestry can offer a sustainable strategy for smallholder systems by enhancing soil quality and the presence of arbuscular mycorrhizal fungi (AMF) in crop roots. Using a canopy-based radial sampling design, we assessed the influence of Mangifera indica (mango) trees on soil properties and AMF communities in maize and cassava in southern Ethiopia. Illumina MiSeq sequencing identified 908 AMF operational taxonomic units (OTUs) from 7 families, dominated by Glomeraceae. While soil properties, including pH, total nitrogen (TN), organic carbon, and potassium, were significantly affected by the distance from mango trunks, this was not the case for AMF community composition and AMF richness and diversity. Host identity, rather than distance from the mango trees, was the primary driver of AMF community composition, with distinct and host-specific assemblages in mango, maize, and cassava roots. Soil nutrients influenced AMF diversity differently across hosts. In maize-mango systems, TN positively affected observed richness (Sobs) and Shannon diversity (N1), whereas Olsen P negatively affected N1 and Simpson diversity (N2). In cassava-mango systems, TN enhanced Sobs, and Olsen P positively influenced expected richness (Sexp). Overall, these findings demonstrate a decoupling between mango-induced soil fertility enhancement and crop AMF community composition and diversity, rather emphasizing the roles of host type and soil nutrients in structuring AMF communities. Without demonstrating direct benefits, we at least show that mango can be effectively integrated into smallholder maize and cassava production without compromising the AMF communities, while enhancing key soil fertility indicators. Maintaining adequate nitrogen levels while avoiding excessive phosphorus inputs may help sustain stable AMF communities in agroforestry systems.

O_LICliffs are environmentally extreme yet biodiversity-rich ecosystems that harbour specialist plants, many endemic and threatened. Plant persistence in these nutrient-poor substrates may depend on tightly linked soil- and root-associated microbial communities, which remain poorly understood. These interactions may become increasingly important with the global expansion of recreational climbing. While physical climbing impacts on vegetation are documented, potential chemical effects, from the use of climbing chalk (magnesium carbonate), on soil properties and plant-associated microbiota remain unknown. C_LIO_LIWe sampled soils and roots beneath cliff-specialist and generalist plants, and unvegetated soils, across climbed and unclimbed routes in northern, central, and southern Spain. Soil physicochemical properties were quantified, fungal communities were characterized using ITS-metabarcoding, and structural equation modelling was used to disentangle direct and indirect effects. C_LIO_LIClimbing increased soil pH and altered soil chemical properties, driving shifts in fungal diversity and functional composition in soil and roots. The relative read abundance of root-associated symbiotrophic fungi declined, whereas arbuscular mycorrhizal fungi and pathogens increased in climbed cliffs. Overall effects were consistent, with cliff-specialist plants mediating nutrient and fungal shifts. C_LIO_LIur findings show that climbing can reshape cliff soil chemistry and fungal communities, with potential cascading consequences for plant functional performance, nutrient dynamics, and ecosystem resilience. C_LI

Streptomyces are abundant soil inhabitants with extensive secondary metabolism and antibiotic resistance traits. Yet, their ecological role in shaping soil antibiotic resistome dynamics remains understudied. Here, we investigated how two different bio-based fertilizers harbouring Streptomyces shaped soil resistome and mobilome by combining genome analysis of eight Streptomyces isolates to metagenomic profiling of soils before fertilization, within 48 hours after fertilizer application, and six weeks after. Streptomyces genomes showed linkages among antibiotic resistance genes, carbohydrate-active enzymes, and antibiotic-production-associated biosynthetic gene clusters, connecting resistance and biosynthesis to broader metabolic strategies. Relationships between carbon degradation and biosynthesis associated with specific enzyme families, indicating that carbon availability shapes secondary metabolism. We confirmed experimentally that antibacterial potential varied with carbon source, suggesting that microbial activity during manufacturing of the bio-based fertilizers may create localized selection pressures before fertilizers enter the soil. Fertilization with the studied materials induced modest but consistent shifts in resistome and mobilome without major changes in dominant taxa or overall bacterial abundances, indicating functional reorganization within soil communities. Diversity of antibiotic resistance genes and mobile genetic elements increased, whereas abundance changes were small. Mobile genetic element composition showed stronger responses that were associated with fertilizer inputs, Streptomyces abundance, and taxa linked to faecal and resistance sources. Together, our results show that bio-based fertilizers shape soil resistome primarily through ecological restructuring of resident soil communities, while carbon-dependent microbial activity within fertilizers may enrich resistance. These factors should be considered in manufacturing of bio-based fertilizer as well as in designing agricultural practices.

Secondary forest succession following agricultural abandonment is a dominant land-use transition across the tropics, yet whether soil microbial communities recover toward old-growth forest reference states remains poorly understood, particularly in West Africa. Here, we investigated the successional dynamics of bacterial and arbuscular mycorrhizal (AM) fungal communities along post-agricultural chronosequences spanning 1 to 43 years across six classified forests in Cote dIvoire, using Bayesian hierarchical models applied to amplicon sequencing data. Both guilds attained moderate to high alpha diversity within the first decade of succession; AM fungal diversity showed moderate evidence of age-related increase thereafter while bacterial diversity showed no directional trend. Pairwise turnover analyses revealed progressive internal convergence in AM fungal communities with plots farther apart in successional time becoming more compositionally similar, while bacterial communities showed only a weak and uncertain tendency in the same direction. Beta-dispersion modelling further indicated progressive within-forest homogenisation of AM fungal communities across abundance-weighted metrics, while bacterial assemblages showed no such stabilisation. Despite this internal convergence, compositional distances to old-growth reference plots remained persistently high for both guilds throughout the chronosequence, with no statistical evidence of recovery toward old-growth states across any dissimilarity metric or guild within the 40-year window. Indicator species analysis identified no robust stage-specific taxa after correction for multiple testing. These results indicate that microbial succession in post-agricultural West African forests is characterised by rapid early reorganisation followed by stabilisation into site-specific assemblages that remain persistently distinct from old-growth reference communities. This outcome challenges the direct application of classical vegetation successional theory to soil microbiomes and suggests that passive regeneration alone is unlikely to restore old-growth microbial communities within restoration-relevant timescales.

Interactions between amoebae and bacteria are increasingly viewed as key drivers of zoonotic pathogen emergence in rodent-dwelling burrows, yet the environmental factors shaping these interactions remain poorly understood. Here, we analyzed soil characteristics and used absolute quantitative high-throughput sequencing to assess microbial communities in active burrow, inactive burrow, and off-burrow soils across four rodent species (marmot, squirrel, gerbil, and vole) in the Hulunbuir grassland of Inner Mongolia, China. This study demonstrates that rodent activity creates chemically distinct soil microhabitats, with nitrate (NO --N) enrichment in active burrow soils consistently observed across rodent species. Elevated soil NO3--N was associated with reduced microbial phylogenetic diversity and reorganization of amoeba-co-occurring bacterial assemblages. Both absolute abundance-based correlations and functional prediction of co-occurring bacteria indicated that amoebae were primarily associated with nitrogen-cycling bacteria in off-burrow soils. In burrow soils, amoebae increasingly interacted with bacterial taxa associated with pathogenicity while retaining ties to nitrogen-cycling taxa. Structural equation modeling and mediation analysis revealed that NO3--N enrichment indirectly linked to increased infectious disease-related functional potential by amoeba-associated bacterial restructuring and coordinated shifts in nitrogen cycling, independent of changes in bacterial abundance. Together, our findings highlight the importance of rodent-driven soil heterogeneity in shaping amoeba-bacteria interactions and suggest that rodent-mediated NO --N enrichment may promote the emergence and persistence of potentially pathogenic bacteria, with broader implications for soil ecosystem functioning and disease-related processes in terrestrial ecosystems.

Soil microbial diversity is shaped by the spatial scale at which communities are sampled, yet standard sampling practices often homogenize samples, obscuring fine-scale spatial structure and diversity patterns. To better understand how sampling effort, spatial extent, and physical homogenization influence plot-level microbial richness estimates, we sampled 57 forest and grassland sites across three regions in Germany using a 14-core cross-transect design and performed 16S rRNA gene metabarcoding. We simulated sampling efforts and a range of spatial extents and compared diversity estimates to those from physically homogenized composite samples. Plot-level richness increased continuously with sampling effort and spatial extent, with no evidence of saturation. However, when sequencing depth was held constant, sampling completeness declined with increasing sampling effort, meaning that more diversity is not captured. Composite samples substantially underestimated plot-level richness and altered apparent diversity relationships between ecosystems; individual cores identified forests as richer than grasslands, whereas homogenized samples suggested the opposite relationship. These results demonstrate that sampling effort, spatial extent, and homogenization fundamentally shape soil microbial diversity estimates. Homogenized composite samples cannot substitute for individual cores when the goal is to reliably quantify plot-level richness or compare diversity across ecosystems.

The contribution of predation between bacteria to microbial community dynamics in agricultural fields has hardly been investigated. Here. dynamics of general prokaryotes (GEP) and of the predators Bdellovibrionales (Bd) and Bacteriovoracales (Bac) (Bdellovibrio-and-Like Organisms, BALOs) were studied in two agricultural fields differing in organic and mineral input regimes, for one year. Season, but not fertilization, affected absolute sizes of GEP and of BALO communities. 16S rRNA gene community analysis identified numerous novel Bd and Bac lineages, with none of the dominant BALOs related to characterized isolates. A few dominant BALO amplicon sequence variants (ASVs) persisted year-round, whereas others showed seasonal- or treatment specific responses. GEP, Bd, and Bac ASV a-diversity was mostly influenced by season, with some changes due to fertilization in Bd, and Bac communities. Seasonal changes, site, and fertilization regimes influenced {beta}-diversity of GEP, Bd and Bac communities and determined the structure of BALO-gram-negative bacteria interaction networks, signaling that niche segregation acts at the microbiome-BALO interface. Accordingly, we suggest that shifts in GEP community structure triggered by environmental changes and agricultural practices cascade to BALO predators, in turn affecting BALO-microbiome interactions. These dynamics may be harnessed to manipulate the soil microbiome to benefit sustainable environmental and agricultural outcomes.

Although biostimulants have attracted attention for sustainable agricultural systems, their efficacy remains poorly understood. In this study, we evaluated the effects of fermented botanical product (FBP) produced by fermenting and aging 41 types of fruits, grains, seaweed, and root vegetables with brown sugar for more than three years. Three crops, tomato, rice, and komatsuna (Brassica rapa), were cultivated with the application of 5,000- or 10,000-fold diluted FBP in greenhouses or fields. Application of diluted FBP promoted plant growth, as indicated by increased fresh weights of shoots, leaves, and roots, fruit production in tomato, and rice husk yield. As diluted FBP contained low nutrient levels, an indirect mechanism of plant growth promotion was suggested. Bacterial community structure analysis indicated changes in alpha diversity, beta diversity, and the predominant phyla in FBP-applied soils without plants and in soils cultivated with tomato, rice, and komatsuna. In addition, the abundance of plant-growth-promoting bacteria, such as Arthrobacter, Pseudomonas, Paraburkholderia, and Planifilum, increased in soils treated with diluted FBP. Furthermore, ammonium formation activity was observed in komatsuna cultivation soils treated with diluted FBP, whereas phosphate-solubilizing activity was enhanced in soils from all three crop cultivation systems treated with diluted FBP. These results suggest that diluted FBP influences bacterial communities and promotes crop growth through indirect effects, including increases in plant-growth-promoting bacteria, ammonium production, and phosphate solubilization. Alternatively, FBP may directly stimulate plant growth. Therefore, FBP may be a useful biostimulant for sustainable agricultural systems. HighlightsO_LIDiluted FBP promoted the growth of tomato, rice, and komatsuna (Brassica rapa). C_LIO_LIDiluted FBP altered the bacterial community structure in cultivated soils. C_LIO_LIFBP increased the abundance of plant-growth-promoting bacteria in cultivated soils. C_LIO_LIFBP stimulated ammonium formation and phosphate solubilization in cultivated soils. C_LIO_LIFBP may be a useful biostimulant for sustainable agricultural systems. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/728655v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4772dorg.highwire.dtl.DTLVardef@12c40e8org.highwire.dtl.DTLVardef@594513org.highwire.dtl.DTLVardef@c5e5a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Climate change is reshaping soil microbial communities, yet the impact of warming in bacterial-fungal interactions (BFIs) remains underexplored. We investigated whether heatwave temperature influence BFIs and the mechanism supporting the interaction. Using co-culture experiments with two bacterial and two fungal strains isolated from heathland soil, we compared mono- and co-cultures final abundances under ambient (18{degrees}C) and heatwave (25{degrees}C) soil temperatures. Our results revealed strongly asymmetric interactions, where fungi benefited by around 5% from bacterial presence, while bacterial abundance was inhibited by around 68%, regardless of temperature. Analyses of pH confirmed that acidification by fungi was probably the main cause of this inhibition. Moreover, warming did not affect the strength or direction of these interactions, though it slightly increased fungal abundance. These findings provide direct experimental evidence that fungi can impact bacteria via acidification, and that the interaction is unaffected by temperature. Understanding these mechanisms is crucial for improving predictions of microbial community dynamics and ecosystem functioning in warming environments.

An agroforestry (AF) system improves crop quality, ecosystem services, and microbial resilience, but its effects on oilseed bioactivity and soil microbiomes are still underexplored. This study compared AF and monocropping systems for castor (Ricinus communis L.) grown in Rajasthan, India, to evaluate plant productivity, seed oil composition, antimicrobial properties, and soil bacterial communities. AF enhanced seed morphology and germination. Castor oil from agroforestry had a 2.4-fold higher phenolic content, 2% more ricinoleic acid, and lower levels of oleic and linoleic acids compared to monocropping, confirmed by infrared spectroscopy and gas chromatography, along with increased expression of the RcDGAT2 gene involved in fatty acid biosynthesis. This led to improved antimicrobial activity against Bacillus mobilis and Pseudomonas fluorescens. Full-length 16S rRNA gene sequencing on the Nanopore platform identified 17 bacterial phyla in soil microbiomes, with Proteobacteria and Firmicutes as the dominant phyla. While alpha diversity was similar, AF soils showed distinct taxonomic shifts, enriching bacteria such as Alkalimonas, Aureimonas, Blastopirellula, Glutamicibacter, Rhizobium, Rhizomicrobium, and Rhodovulum, linked to nutrient cycling and plant growth promotion. Isolated rhizospheric/root endophytic Bacillus safensis and Enterobacter cloacae from AF castor exhibited plant growth-promoting traits via biochemical tests and whole-genome sequencing; their oil biosynthesis genes likely contribute to host oil quality by enhancing precursor supply and phenolic pathways. These isolates enhanced the growth of the model plant Arabidopsis thaliana. In summary, AF enhances the bioactivity of castor oil and microbial functions by modulating plant-soil-microbe interactions, thereby supporting sustainable crop quality and soil health.

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.

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.