Metabolic engineering of crops can redirect host carbon flux, but its consequences for microbiomes remain unclear. Here, we show that engineering oilcane for triacylglycerol (TAG) accumulation reshapes rhizosphere microbial guilds across greenhouse and field environments while preserving functional capacity. Using 36 rhizosphere metagenomes from wild-type sugarcane and engineered oilcane accessions, we reconstructed metagenome-assembled genomes and linked community turnover with shifts in functional potential. Oilcane rhizospheres exhibited taxonomic restructuring relative to wild-type plants, driven primarily by turnover rather than nestedness and marked by genotype-dependent replacement of microbial guilds. These patterns were strongest in accession 1566 and amplified under field conditions. Despite these compositional shifts, broad patterns of functional potential remained similarly distributed, whereas pathway-level differences were evident in energy production and conversion, lipid transport and metabolism, secondary metabolite biosynthesis, transport and catabolism, and signal transduction. These findings extend evaluation of engineered crops beyond host traits alone to include microbiome-scale responses.

Coniferous forest soils represent a globally important carbon sink, where the microbiome is essential for carbon flux between tree roots, rhizosphere, litter and soil. Soil habitats, such as roots, rhizosphere, bulk soil and litter differ in physicochemical properties and composition of highly specialized microbial communities, whose activity reflects the seasonality of temperature and tree activity of these mid- to high-latitude biomes. Here we present a multi-omic dataset encompassing 160 samples collected from four coniferous forest soil habitats in the Czech Republic and Norway, sampled in early summer, late summer, early winter and late winter that characterize the composition, genomic potential and activity of tree roots and microbiome. For each sample, we provide metabarcoding-based composition of bacterial, fungal and eukaryotic communities, results of shotgun DNA sequencing (metagenomes) and shotgun RNA sequencing (metatranscriptomes) illustrating the functional potential and activity within habitats. This dataset enables analyses of the temporal variation of taxonomic composition, functional potential and transcription across seasons in a temperate and boreal coniferous forest.

Abstract Wastewater treatment plants (WWTPs) are known reservoirs of antibiotic resistance genes (ARGs). Non-antibiotic compounds such as antidepressants may further promote ARG acquisition through horizontal gene transfer (HGT). Desvenlafaxine, a serotonin-norepinephrine reuptake inhibitor (SNRI) listed on the EU Surface Water Watch Lists, is among the most frequently detected antidepressants in WWTP effluents, yet its role in HGT has not been examined. Here, we detected desvenlafaxine at the highest concentrations among four antidepressants monitored across three municipal WWTPs in western New York. Using Acinetobacter baylyi ADP1 as a model recipient in natural transformation assays (n = 6), we found that desvenlafaxine significantly increased transformation frequency at 10 mg/L (1.74 ± 0.33-fold) and 50 mg/L (1.49 ± 0.19-fold; P adj < 0.05). Effects were independent of reactive oxygen species or membrane permeability stress, consistent with its very low toxicity (IC 20 ∼1353 mg/L). Instead, desvenlafaxine induced dose-dependent increases in membrane fluidity and shifts to less negative zeta potentials, suggesting that electrostatic interactions between its cationic amine group and the negatively charged membrane reduce surface repulsion and facilitate plasmid proximity during uptake. Non-targeted proteomics revealed a biphasic response: at 10 mg/L, competence-associated proteins (PilB, ComM) were upregulated and STRING analysis identified networks linked to membrane transport, transcriptional regulation, and envelope remodeling, while no connected network was recovered at 50 mg/L. Electron microscopy confirmed higher pili frequency at both doses. Together, these findings reveal an overlooked role of this non-antibiotic pharmaceutical in promoting ARG spread from wastewater environments. Importance The spread of antibiotic resistance poses a serious and escalating threat to human health worldwide. While antibiotic use is widely recognized as a key driver, non-antibiotic pharmaceuticals released into the environment through wastewater have received far less attention. Antidepressants are among the most frequently detected drugs in treated wastewater effluents, yet their potential to promote antibiotic resistance transfer in bacteria remains poorly understood. This study demonstrates that desvenlafaxine, one of the most abundant antidepressants found in municipal wastewater, increases the uptake of antibiotic resistance genes in environmental bacteria and identifies the bacterial cell-surface changes that enable this.

Coral-associated microbial communities play a critical role in the health and resilience of reef ecosystems; however, the relative importance of host identity and environmental factors in shaping these communities remains unclear, particularly in understudied regions such as the Gulf of California. In this study, we characterized the taxonomic composition, diversity patterns, persistent taxa (core bacteriome), and predicted functional potential of bacterial communities associated with three coral genera (Pocillopora, Porites, and Pavona) and surrounding seawater using 16S rRNA gene amplicon sequencing and PICRUSt2-based functional inference. Bacterial community structure differed significantly among coral hosts (PERMANOVA, p < 0.01), whereas geographic location and measured physicochemical parameters had no detectable effect. Coral-associated bacterial communities exhibited lower alpha diversity than seawater and formed distinct host-specific clusters in beta-diversity analyses. Core bacteriome analysis revealed a combination of conserved and host-specific taxa, with Acinetobacter consistently present across hosts, while genera such as Pseudovibrio and Ruegeria showed host-specific associations. Differential abundance analyses further confirmed distinct bacterial signatures among coral genera. Predicted functional profiles were dominated by central metabolic pathways and exhibited significant differences among hosts, although overall functional composition remained relatively conserved. Stratified analyses indicated that similar metabolic pathways were supported by different taxonomic assemblages, suggesting functional redundancy. Overall, our results demonstrate that host identity is the primary driver of both taxonomic composition and predicted functional potential in coral-associated bacterial communities in the Gulf of California, highlighting the coexistence of stability and host-specific differentiation within the coral holobiont.

Soil is the largest active terrestrial carbon reservoir. However, modeling soil carbon decomposition across diverse environments remains challenging, as most models use uniform, predefined parameter values within the same biome or soil type. Here, using a continental-scale incubation dataset of 156 soil samples from 20 National Ecological Observatory Network sites and a mechanistic model, we quantified decomposition parameters—decay rate (K) and carbon use efficiency (CUE)—for particulate and mineral-associated organic carbon across the US. Notably, K and CUE exhibited a 10-fold variation across soils, influenced by well-recognized (pH and nitrogen) and underrepresented geochemical and microbial factors (e.g., oxalate-extractable iron, fungal abundance). By integrating key predictors, we generated gridded maps of K and CUE, unveiling different spatial decomposability patterns of particulate and mineral-associated organic carbon across the contiguous US. Our findings underscore the need to incorporate underrepresented edaphic factors into models to improve spatially heterogeneous carbon decomposition estimations.

Identifying core taxa in microbial ecology highlights groups likely to participate in a broad range of potential ecological interactions. Here, we present BRCore, an R package to identify core taxa using abundance-occupancy distributions and beta-diversity contributions across ecological niches, and predict stochastic and deterministic taxa.

The perennial grass Miscanthus × giganteus (miscanthus) offers a sustainable alternative to traditional biomass feedstocks while improving key soil health parameters, including aggregation. Aggregate stability results from dynamic soil-plant-microbe interactions, yet the relative importance of each factor remains an active research question. Building on previous observations that miscanthus alters soil structure to improve water-holding capacity and aggregate stability, we characterized the communities of soil bacteria and arbuscular mycorrhizal fungi (AMF) across three sites in Iowa, USA, comparing miscanthus to annual maize ( Zea mays L.) and non-cropped perennial turfgrass ( Poa spp.). We examined whether microbiomes co-varied with soil aggregation and, if so, whether plant cover identity or life history categorization better explained the observed patterns. Bacterial and AMF communities varied across sites and plant types, with signals that life history and plant cover identity both mattered. Aggregate stability aligned with a perennial-annual divergence in microbial beta diversity, while finer-scale differences in community composition and network structure were plant-specific. Soils under perennial plants were enriched in microbial groups positively correlated with aggregate stability; we identified 61 bacterial and 8 AMF "architect" taxa for future study. Within- and cross-kingdom co-occurrence network analysis revealed greater complexity under perennial plants: 1.9-fold more network links in miscanthus bacteria-bacteria networks than in maize, and 1.7-fold more in turfgrass AMF-AMF networks. Miscanthus fundamentally shapes microbial interactions, particularly among bacteria, relating to improved soil physical structure. Understanding these soil-plant-microbe feedbacks advances the development of biomass feedstocks with a portfolio of soil health benefits for next-generation biofuels and bioproducts.

Dairy manure reception pits receive fresh manure and milkhouse wash water, and have short residence times, yet their role as reservoirs of extracellular antibiotic resistance genes (exARGs) remains poorly characterized. Here, we quantified six ARGs (tetG, tetM, tetX-tetracycline, sul1-sulfonamide, and ermB-macrolide) and three integron-associated mobile genetic elements (MGEs: intI1, intI2, and intI3) in intracellular DNA (iDNA) and extracellular DNA (exDNA) from fresh manure and from a dairy reception pit with an estimated residence time of <6 h. While total DNA yields were lower in the pit relative to fresh manure samples, exDNA-to-iDNA ratios were significantly higher for all genes (P < 0.001), indicating rapid enrichment of exDNA after manure entered the pit and mixed with the wash water. Notably, tetM exhibited a higher free (unattached) to bound (surface-attached) exDNA ratio in pit samples, which suggested greater accessibility to this gene within the extracellular pool in the pit. Exploratory correlation analysis identified recurring ARG-MGE associations, whereas 16S rRNA gene profiling showed iDNA-associated taxonomic composition diverged between fresh and pit samples, while exDNA-associated taxonomic profiles remained more similar. This result could support that exDNA is relatively stable over time and in varying environments, and that iDNA is more reflective of selective pressures. Together, these results indicate that even short-term reception pits can act as reservoirs of exDNA-associated resistance determinants and justify further work on their persistence, mobility, and management in dairy systems. IMPORTANCE To date, extracellular DNA (exDNA) has been shown to contribute to the spread of antibiotic resistance genes (ARGs) in the environment; however, few studies have evaluated its enrichment in short-term dairy pit-stored manure systems. This study demonstrates that dairy manure pits concentrate exDNA during short-term storage and serve as reservoirs for ARGs, along with mobile genetic elements that can facilitate subsequent gene transfer. The results of this study are a strong rationale for further investigation and targeted management strategies of exDNA in manure pits.

The concept of a core microbiome emerged from host-associated research to describe microbial members or functions conserved across clearly defined spatial, temporal, and biological boundaries. In soil- and plant-associated microbiome research, however, the term has increasingly shifted toward analytically defined subsets selected using study-specific thresholds or criteria. Synthesizing recent literature and cross-site analyses of bioenergy crop field soils, we show that the original biological meaning of the core microbiome has been blurred by dataset-specific analytical criteria. Taxa designated as 'core' were highly sensitive to methodological choices and often reflected explanatory value rather than conserved biological membership. Moreover, many studies that identify taxonomic 'core' members interpret their significance in functional terms, suggesting that functional conservation may be the biological interest. Taxonomic conservation may not be the most biologically meaningful target in highly heterogeneous soil and rhizosphere systems, where functional conservation may persist despite taxonomic turnover. Accordingly, 'core microbiome' should be reserved for microbial components explicitly demonstrated to be conserved across defined spatial, temporal, and environmental dimensions and linked to conserved ecological functions, while taxa selected for explanatory value are better described as 'explanatory subsets of taxa'. Greater terminological precision will improve cross-study comparability and strengthen ecological inference in plant-soil microbiome research.

Background and AimsPerennial crops often improve soil structure relative to annual systems, but the biochemical pathways by which plants influence soil aggregation remain unclear. We tested whether seven years of Miscanthus × giganteus cultivation improved soil aggregation relative to continuous maize and whether these changes were associated with microbial- versus plant-derived biochemical compounds.MethodsWe sampled soils from three Iowa sites under maize, miscanthus, and adjacent turfgrass alleyways as a perennial reference. We measured water-stable aggregates, mean weight diameter, microbial biomass, extracellular polysaccharides, amino compounds, and extractable carbohydrates, including the microbial-to-plant carbohydrate monomer ratio (GM:AX).ResultsMiscanthus increased water-stable aggregates and mean weight diameter by ~41% and ~74%, respectively, relative to maize across sites, while turfgrass showed smaller increases (36% and 61%). Contrary to our second hypothesis, microbial-derived pools, including microbial biomass, extracellular polysaccharides, and amino compounds, showed limited differences among treatments. Instead, miscanthus uniquely altered extractable carbohydrate composition, decreasing GM:AX by ~11% relative to maize and turfgrass, indicating a stronger contribution of plant-derived carbohydrates to the labile carbon pool. In contrast, turfgrass increased aggregation alongside higher microbial biomass and amino compounds, consistent with a more microbially mediated pathway.ConclusionThese results show that perennial plants can improve soil aggregation through distinct biochemical pathways at the plant–soil interface. Miscanthus was associated primarily with shifts in plant-derived carbohydrates, whereas turfgrass showed stronger microbial signatures. This work advances mechanistic understanding of how perennial vegetation shapes soil structure and may help guide the design of cropping systems that improve soil function.

Quantifying the carbon (C) uptake of Miscanthus x giganteus (M x g) in both aboveground and belowground structures (e.g., net primary productivity (NPP)) and differences among methodological approaches is crucial. Our objectives were to directly measure Mxg NPP and evaluate the effects of nitrogen application, location, and belowground biomass sampling methods. We hypothesize that increased nitrogen application increases the overall NPP of M x g and that quantifying rhizome biomass using excavations will produce the lowest variability between replicates. We collected biomass from mature M x g stands from three locations in Iowa with three nitrogen application rates and one site in Illinois. We destructively sampled at two time points, when rhizome mass is anticipated to be at a minimum (initial) and anticipated to be at its maximum (peak). Biomass was collected from 1 x 1 m quadrats in which one in-clump and one beside-clump cores were collected and then excavated to 30 cm depth to extract all rhizomes. We found that aboveground M x g NPP ranged from 15.4 Mg Da ha-1 year-1 to 36.4 Mg Da ha-1 year-1 and belowground M x g NPP ranged from 4.4 Mg Da ha-1 year-1 to 19.6 Mg Da ha-1 year-1. M x g NPP varied across sites, fertilization, and calculation assumptions. Aboveground NPP (yield) was on average 68.7% of the total NPP. Root-to-shoot ratios at peak biomass decreased with nitrogen application rate, from an average of 1.9 for 0 N plots to 0.89 for 224 N fertilized plots. There was more variation in core data than from excavations; however, when in-clump and beside-clump cores were averaged together, core and excavation averages were not different. Overall, these results show that the range of mature M x g NPP is driven by aboveground productivity, influenced by nitrogen application and site. Our results provide useful data to constrain agro-ecosystem models and provide crucial insights for future perennial belowground sampling.

The sustainable production of purpose-grown bioenergy feedstocks is essential in transitioning away from fossil fuels. Synthetic communities (SynComs) are consortia of microorganisms that can be used as biological interventions to support objectives like plant growth and stress tolerance. This review examines the state of knowledge regarding microbiomes and SynComs of second-generation bioenergy feedstocks, focusing on the rhizosphere. We first provide an overview of second-generation feedstocks, including switchgrass (Panicum virgatum), miscanthus (Miscanthus × giganteus), sorghum (Sorghum spp.), sugarcane (Saccharum spp.), and poplar (Populus spp.), and summarize our current understanding of their plant-soil-microbiome ecology. We next discuss considerations in the objectives, design, and evaluation of SynComs to enhance feedstock production, and then critically review the literature around their use. Our literature analysis revealed that SynCom performance varied substantially between controlled pilot experiments and field trials, possibly due to system complexity that could not be fully considered in their design and pilot evaluation. We identified a gap in the use of SynComs to support the unique sustainability objectives of biofuel feedstock agriculture, presenting an opportunity to leverage these additional microbial traits in SynCom designs. Finally, we emphasize the importance of targeted research to identify the ecological principles that govern the assembly, activation, and persistence of microbes in the feedstock rhizosphere, thereby enhancing our capacity to manage microbiomes under diverse environmental conditions and ensure their functionality. Beyond biofuels, SynComs are a promising microbiome management strategy for crop production; however, an ecologically informed design and evaluation of SynComs are advised.

Individual fungi and bacteria can decompose lignin, but little is known about how specific taxa and their interactions may be related to this critical carbon-cycling process across diverse environments. We characterized relationships between bacterial and fungal communities and mineralization of isotope-labeled lignin across 156 incubated mineral soil samples collected from 20 National Ecological Observatory Network sites spanning diverse ecosystems (tundra to tropics) across North America. Based on marker gene sequencing, bacteria were more closely related to lignin mineralization than fungi at the levels of overall community composition, individual taxa, and co-occurrence network. We identified 14 bacterial and fungal genera across sites and 26 taxa (mostly bacteria) within sites, including two genera (Occallatibacter and Terracidiphilus) that were significantly related to lignin mineralization within and across sites. Additionally, many microbial ‘modules’ from co-occurrence networks were related to lignin mineralization, and this was even more evident during the later stages of decomposition. This suggests the importance of microbial interactions for lignin decay and implies that microbes interacted in a way favoring lignin decomposition over the incubation. We identified 10 bacterial-fungal interactions (BFI) that could significantly strengthen and 10 BFI that could weaken microbial relationships with lignin mineralization, indicating that synergistic and antagonistic BFI were both important. Overall, our study illustrated the key importance of microbial interactions even more so than individual taxa for predicting lignin mineralization.

ABSTRACTNutrient inputs influence the sustainability of bioenergy crop production through contemporary (shortly after addition) and legacy effects (persisting over years) on microbial nitrogen (N) and carbon cycling, which contribute to greenhouse gas emissions. However, the relative importance of contemporary and legacy effects and how that could vary by crop functional types is poorly understood. Considering its rhizomatous roots and perennial growth, we hypothesized that Miscanthus × giganteus (M×g) would be more sensitive to legacy N fertilization and the historical context of its environment than an annual crop like maize. To test this hypothesis, we examined the effects of legacy and contemporary N inputs on nitrous oxide (N2O) and carbon dioxide (CO2) emissions, as well as key N cycling genes in soils where M×g and maize were grown. A 150‐day soil incubation experiment was conducted using soils from a long‐term M×g and maize fertility experiment with three historic N fertilization rates (0, 112, and 336 kg N ha−1 year−1) and a contemporary amendment (60 mg N kg−1) with negative control (0 mg N kg−1). We observed significant increases in cumulative N2O emissions in Mxg soils relative to maize soils, particularly at higher legacy fertilization rates, while contemporary N had no significant effect. Bacterial amoA gene abundance, which plays a significant role in nitrification in nutrient‐rich soils, also increased with higher legacy fertilization rates in M×g soils but was unaffected by the contemporary N. In maize soils, legacy and contemporary N did not significantly affect N2O emissions, but cumulative CO2 emissions and amoA gene abundance significantly increased. The abundances of norB genes were not significantly influenced by either legacy fertilization or contemporary N amendments in either soil. Our findings demonstrate the greater importance of fertilization history over contemporary N in mediating soil N2O emissions, particularly for perennial bioenergy crops.

Swine manure is typically applied to agricultural land at agronomic rates, providing essential nutrients to crops and enhancing soil fertility. While providing nutrients, manure can also introduce pathogens and other contaminants, including antibiotic residues and antibiotic-resistant bacteria (ARB) and genes (ARGs), into the environment with subsequent threats of transfer to humans through the food chain. There are various manure management strategies that may be considered before land application to potentially mitigate the risks of antimicrobial resistance exposure. In a laboratory manure slurry incubation study, the effectiveness of five different manure treatment practices was evaluated. These practices included aeration, anaerobic digestion (AD), heat, liming, and solid-liquid separation (SLS), and were assessed for their ability to reduce antibiotics, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs). This study focused primarily on the impacts on tetracycline (TET), TET-resistant populations, and TET-resistant genes. Additionally, we evaluated the impacts of manure treatments on macrolide (MAC) resistance. We compared the effects of manure treatment on phenotypic resistance of TET, the concentration of TET and MAC residues, and copies of several TET- and MAC-resistant genes. For TET-resistant ARB, our results indicate that AD and liming are the most effective among all treatments. Liming was able to rapidly reduce TET-resistant ARB. Comparing ARGs and antibiotic residuals, we found AD to be the most effective treatment in reducing TET-resistant genes (tetO, tetM, and ermB) and total TET and MAC residues. This research provides valuable insights that can be used to recommend best management practices to farmers and policymakers to reduce dissemination of antimicrobial resistance through environmental pathways.

Bacteria in the honeybee gut are a well-recognized factor affecting bee health. However, the primary focus of this research has been the hindgut, while the crop, or honey stomach, is assumed to be dominated by environmentally acquired transient taxa that matter little to the bees. To evaluate this assumption, we examined bacterial taxa in the crop and mouth of Apis mellifera and A. cerana japonica foragers and in the nectar of Prunus mume flowers visited by the bees in the Minabe-Tanabe region of Japan. We found that in bacterial composition, the crop was distinct from both the mouth and the nectar, whereas mouth and nectar samples were indistinguishable. Furthermore, the crop remained similar in bacterial composition and diversity, while the mouth showed a sharp drop in alpha diversity and a large increase in beta diversity, from summer to winter. These results refute the conventional assumption, suggesting instead that the crop contains a conserved bacterial community largely distinct from environmental taxa. We also found that strains of a crop-associated species, Apilactobacillus kunkeei, could be season- and host species-specific. Together, these findings suggest that crop-associated bacterial communities should be studied further to better understand the relationship between honeybees and their gut bacteria.

Dairy manure pit storage systems are significant reservoirs for antimicrobial resistance genes (ARGs). These genes occur in both intracellular DNA (iDNA) and extracellular DNA (exDNA), but their distribution across these categories in fresh (loafing pen surface) and pit-stored dairy manure has not been previously characterized. To address this gap, we quantified the abundance of six ARGs ( tetG , tetM , tetX- tetracyline, sul1 -sulfonamide, and ermB- macrolide) and three mobile genetic elements (MGEs) ( intI1 , intI2 , and intI3 ) in iDNA and exDNA extracted from fresh and pit-stored manure collected at a dairy farm in Iowa. While total DNA yields were lower in pit-stored relative to fresh manure samples, exDNA-to-iDNA ratios were significantly elevated across all genes (p<0.001), indicating relative enrichment of exDNA during storage. Notably, tetM exhibited a higher free (unattached) to bound (surface-attached) exDNA ratio in pit samples, which suggested an increased potential for gene transfer in the pit. Correlation network analysis revealed similar numbers of strong ARG–MGE associations in pit and fresh exDNA, but lower interconnectivity in pit exDNA. Merging fresh and pit datasets showed wider ARG-MGE associations: intI1 and intI3 strongly co-occurred with tetracyclines and macrolide resistance in iDNA, while sul1 correlated with MGEs only in the exDNA network. Microbial community profiling showed similar taxa in exDNA across manure types, while iDNA communities diverged significantly. This result could support that exDNA is relatively stable over time and in varying environments, and that iDNA is relatively more reflective of selective pressures. Overall, our results highlight exDNA as a critical but overlooked reservoir of resistance determinants, warranting further investigation and targeted management strategies in dairy systems. Importance To date, extracellular DNA (exDNA) has been shown to contribute to the spread of antibiotic resistance genes (ARGs) in the environment; however, few studies have evaluated its enrichment in dairy pit-stored manure systems. This study demonstrates that dairy manure pits concentrate exDNA during dairy manure storage and serve as a reservoir for ARGs, along with mobile genetic elements that can facilitate subsequent gene transfer. The results of this study are a strong rationale for further investigation and targeted management strategies of exDNA in manure pits.

Soil is one of the largest reservoirs of microbial diversity in nature. Although soil management is vital for agricultural purposes, intensive practices can have a significant impact on fertility, microbial community, and resistome. Thus, the aim of this study was to evaluate the effects of an intensive soil management system on the chemical attributes, composition and structure of prevalent bacterial communities, and presence and abundance of antimicrobial resistance genes (ARGs). The chemical characterization, bacterial diversity and relative abundance of ARGs were evaluated in soils from areas of intensive vegetable cultivation and forests. Results indicate that levels of nutrients and heavy metals were higher in soil samples from cultivated areas. Similarly, greater enrichment and diversity of bacterial genera was detected in agricultural areas. Of the 18 target ARGs evaluated, seven were detected in studied soils. The oprD gene exhibited the highest abundance among the studied genes and was the only one that showed a significantly different prevalence between areas. The oprD gene was identified only from soil of the cultivated areas. The blaSFO, erm(36), oprD and van genes, in addition to the pH, showed greater correlation with in soil of cultivated areas, which in turn exhibited higher contents of nutrients. Thus, in addition to changes in chemical attributes and in the microbial community of the soil, intensive agricultural cultivation systems cause a modification of its resistome, reinforcing the importance of the study of antimicrobial resistance in a One Health approach.