Exploring the Role of Root Exudates in Shaping Plant–Soil–Microbe Interactions to Support Agroecosystem Resilience

Abstract

Root exudates are key mediators of plant–soil–microbe interactions, shaping rhizosphere dynamics and influencing agroecosystem resilience. Comprising diverse primary and secondary metabolites, these compounds are actively secreted through specific transport pathways and are modulated by intrinsic plant traits and environmental conditions. Root exudates serve as chemical signals that recruit and structure microbial communities, facilitating nutrient mobilization, microbial feedbacks, and the regulation of plant growth and stress responses. By modulating soil chemical, physical, and biological properties, exudates contribute to carbon cycling, soil health, and the maintenance of ecosystem services. Moreover, they play multifunctional roles in enhancing plant tolerance to abiotic and biotic stresses, while also mediating interactions with neighboring plants. This review provides a holistic perspective on root exudation, encompassing their mechanisms and drivers, roles in rhizosphere ecology and plant stress adaptation, and methodological advances, while highlighting opportunities to harness these processes for resilient, productive, and sustainable agroecosystems.

1. Introduction

Global climate change is recognized as a major threat to the natural ecosystems [1]. It is a complex process involving shifts in environmental conditions, including an increased frequency of extreme weather events such as heatwaves, droughts, intense rainfall, floods, and altered freezing patterns [2]. This phenomenon is expected to intensify abiotic stresses (e.g., drought, salinity, nutrient limitation, temperature extremes) and biotic stresses (e.g., pests, pathogens, and invasive species) on crops, thereby reducing the profitability and sustainability of agricultural production systems [3]. Therefore, enhancing plant resilience to stress has become essential to sustain agricultural productivity under the ongoing pressures of climate change and the growing global population. Plants have evolved a broad spectrum of adaptive and defensive mechanisms, including physiological and biochemical adjustments that enable them to cope with environmental challenges [4].

Roots play an essential role in these adaptive processes. As the primary interface between plants and the soil environment, they are the first organs to perceive and respond to fluctuations in soil moisture, temperature, salinity, and nutrient availability [1]. Beyond their role in resource uptake and anchorage, roots actively influence the surrounding soil environment through the secretion of exudates, which mediate plant–soil–microbe interactions. These compounds are increasingly recognized as key drivers of plant adaptation, shaping rhizosphere dynamics and enhancing resilience [5,6]. These organic compounds comprise a complex mixture of primary (carbohydrates, amino acids, and organic acids) and secondary metabolites (phenolics, terpenoids, alkaloids, fatty acids, and phytohormones) [7,8]. The release of these signaling compounds establishes a direct communication pathway between plants and rhizosphere microbes, influencing soil properties, water availability, and plant performance, ultimately enhancing plant tolerance and survival under stress conditions [9]. Thus, plants, soil, and climate form a highly interconnected system, whose complexity is further intensified by the impacts of climate change [10].

Although root exudates have been extensively studied, existing reviews mainly focus on isolated components of the system, such as exudate chemistry, microbial recruitment, or stress-specific responses. In this context, the present review aims to provide a comprehensive synthesis and critical overview of current knowledge on the role of root exudates in promoting plant resilience and sustainability under the pressures of climate change. By integrating evidence across molecular, physiological, ecological, and methodological dimensions, this review advances a framework that positions root exudates as central mediators of plant–soil–microbe interactions. In addition, this review aims to identify knowledge gaps and application-oriented perspectives to guide future research and support the strategic management of root exudate dynamics for the development of resilient and sustainable agroecosystems.

2. Root Exudation: Composition, Transport Pathways, and Intrinsic Drivers

2.1. Definition and Classification of Root Exudates

Root exudates are a diverse array of bioactive metabolites released by plant roots into the surrounding soil, where they play a pivotal role in sustaining plant development and mediating interactions with both physicochemical and biological components of the rhizosphere [11]. The quantity and composition of exudates are highly dynamic and shaped by an interplay of factors, including species and genotype, developmental stage, soil physicochemical properties, environmental conditions, and the diversity of surrounding microorganisms [11,12,13]. Because of this specificity, root exudates act as chemical attractants or repellents that strongly influence the assembly of rhizosphere microbial communities [13]. Even small alterations in exudation patterns induced by biotic or abiotic stress can significantly affect the abundance, activity, and composition of plant-associated microorganisms [14,15].

Beyond mediating biotic interactions, root exudates influence key soil physicochemical properties, including pH, texture, structure, and mineralogy, as well as enhance nutrient cycling and uptake. They contribute to the acquisition of nutrients (e.g., Fe, P, N), enhance tolerance to Al toxicity, improve resource-use efficiency and function in allelopathy. Root-derived compounds also act as signaling molecules that attract symbiotic partners, such as rhizobia or arbuscular mycorrhizal fungi (AMF), and promote beneficial colonization by rhizobacteria [11,14,16].

2.1.1. Primary and Secondary Metabolites and Functional Roles

Primary metabolites include organic acids, amino acids, and carbohydrates. Among their best-characterized ecological roles is the mobilization of soil-bound nutrients by converting unavailable into soluble, plant-accessible forms [14,16].

Amino acids contribute to stress signaling (e.g., histidine, β-alanine, lysine, phenylalanine), stress mediation (e.g., arginine, methionine, serine, proline) and mitigation (e.g., methionine—ethylene precursor/auxin cofactor; proline—RuBisCO protection; serine—homeostasis maintenance), and nutrient acquisition or transport (e.g., asparagine, cysteine, α-alanine, glycine) [17,18,19]. Certain amino acids additionally function as chemo-signals that attract beneficial microorganisms (e.g., glutamine, proline) [20,21].

Organic acids such as citric, malic, and fumaric acid chelate metal ions and modulate soil pH and mineral solubility, thereby improving nutrient availability [22]. They also act as chemoattractants, facilitating the colonization of plant growth-promoting rhizobacteria (PGPR) such as Bacillus sp. [23]. Citric and tartaric acid help mobilize Fe and S as well as support phytoremediation processes [16,24,25]. Lactic acid recruits beneficial lactic-acid bacteria (LAB) [26], whereas aspartic acid contributes to N assimilation [27].

Sugars serve as immediate energy sources for rhizosphere microbes and influence community composition [14]. Xylose is typically released during early root development through the breakdown of hemicellulose [28]. Glucose and fructose, together with sucrose, are major components of root mucilage, which enhances soil aggregation, water retention, and microbial habitat quality [16,29].

Secondary metabolites, including terpenoids, alkaloids and phenolic compounds, constitute a rich chemical arsenal that shapes rhizosphere ecology through microbial modulation, allelopathy and niche defense [14]. Flavonoids regulate microbial recruitment, as shown for quercetin, which enhances arbuscular mycorrhizal colonization under herbivory [30]. Terpenoids serve as antimicrobial, antifungal, and antioxidant agents and can act as biochemical pesticides, as shown for D-limonene, estragole, and related volatiles that inhibit Phytophthora capsica through disruption of zoospore behaviour and ROS induction [31]. Other terpenoids, including isoprene, modulate plant–microbe interactions [32]. Phenolics such as vanillin suppress nematode parasitism [33], and alkaloids like nicotine play central roles in defense against herbivory [34]. Secondary metabolites also mediate allelopathic interactions that suppress neighboring plants or parasitic species [13,35].

2.1.2. Categorization by Molecular Weight and Composition

Root exudates are broadly classified into Low-Molecular-Weight Compounds (LMWCs) and High Molecular Weight Compounds (HMWCs) [12].

LMWCs dominate the rhizosphere both in diversity and abundance. They include simple sugars (e.g., glucose, fructose, xylose), amino acids, inorganic ions (including H⁺), and a wide array of organic acids. Some secondary metabolites, such as sorgoleone, flavonoids and coumarins, are also part of this fraction [11,12,16,36].

HMWCs comprise polysaccharides (e.g., cellulose, starch), proteins, which includes enzymes involved in nutrient mobilization (e.g., phosphatases, peptidases), as well as phenolics such as tannins and mucilage [11,37].

A mechanistic understanding of how these compounds are released requires examining the cellular transport pathways and regulatory processes underlying exudation.

2.2. Mechanisms of Exudation and Transport Across Root Tissues

There are multiple pathways and membrane transport proteins responsible for moving organic compounds and specialized metabolites within root cells and from the cytosol to the rhizosphere [38]. The specific route taken by each compound is determined largely by its physicochemical properties.

The plasma membrane restricts the passive diffusion of large uncharged polar molecules (e.g., glucose) and all charged solutes, while permitting small non-polar molecules and gases to cross the lipid bilayer [39]. Consequently, root exudation requires the controlled movement of metabolites from the cytoplasm, across the plasma membrane, and into the apoplast and soil matrix. This mobilization relies on passive and active transport mechanisms that together govern the efflux, retention, or reuptake of rhizosphere-bound compounds.

Passive transport enables the movement of solutes down concentration or electrochemical gradients without direct ATP expenditure, underpinning the efflux of many LMWCs [6]. This includes simple diffusion of small non-polar molecules and facilitated diffusion through channels or permeases for larger polar molecules and small ions (e.g., Na⁺, K⁺, Cl⁻), crucial for maintaining cellular homeostasis and osmotic status [6,40]. Transporters such as SWEET proteins (sugar efflux) and UMAMIT carriers (amino acid transport) exemplify facilitated diffusion systems involved in exudation [6,12]. Although passive transport itself is energetically “cost-free”, plants exert significant regulatory control through energy-dependent processes, including secondary active reuptake of metabolites, transcriptional and post-translational regulation of transporters, vacuolar sequestration, and modulation of meristematic activity [40].

Active transport, by contrast, requires metabolic energy to move substrates against their gradients, enabling plants to fine-tune the chemical composition of the rhizosphere in response to environmental signals [6,40]. This mode is essential for the selective high rates of efflux of carboxylates (e.g., citrate, malate, oxalate) and many secondary metabolites [41]. Several families of membrane transport proteins (MTPs) mediate active exudation, including the following [38,40,41,42]: The ATP-Binding Cassette (ABC) transporters, primary active pumps that export a broad range of substrates (metabolic products, ions, lipids, and xenobiotics) using ATP hydrolysis; Secondary active transporters, which use the electrochemical gradient of other ions (H⁺ ou Na⁺) to energize substrate efflux, such as the Multidrug and Toxic Compound Extrusion (MATE) family (exporting phenolics, alkaloids and organic acids like citrate), the Aluminum-Activated Malate Transporters (ALMTs), which secrete malate to chelate toxic Al or mobilize nutrients, and the Major Facilitator Superfamily (MFS) carriers, some of which mediate the transport of specialized metabolites including phytosiderophores.

Finally, the secretion of HMWCs, occurs predominantly via vesicular trafficking. This exocytotic pathway requires energy for vesicle formation, trafficking and fusion with the plasma membrane, representing a key route for the release of macromolecules into the rhizosphere [38].

2.3. Plant Characteristics Influence on Exudation Patterns

The quantity and chemical profile of root exudates are highly plastic and dynamic, regulated by a complex interplay between intrinsic plant characteristics (genotype and developmental stage) and extrinsic biological and environmental conditions.

Numerous studies demonstrate that exudation profiles vary substantially among plant families, species, and even cultivars grown under similar conditions [43,44,45,46]. However, the relationship between plant phylogeny and exudate chemistry is complex, and no universal or linear correlation has been identified across plant groups [43,44,45,46]. This specificity highlights the potential for selecting or breeding cultivars with exudation patterns optimized for particular soil or microbial interactions.

Developmental stage and growth rate strongly influence root exudation. Exudate composition is highly dynamic throughout the plant life cycle, with clear temporal shifts as plants transition from seedlings to vegetative, flowering, and reproductive stages, resulting in corresponding changes in the rhizosphere metabolome [46]. Total organic carbon (TOC) exudation generally peaks during early development and declines with plant age [47,48]. These changes are closely linked to primary metabolism: higher photosynthetic activity in young plants provides substrates for rhizodeposition, supporting rapid growth, whereas reduced photosynthesis in mature plants lowers exudation and soil C allocation [49,50,51].

A compositional shift is also observed: sugars and organic acids typically predominate during early developmental stages, often supporting rapid microbial recruitment and initial rhizosphere assembly, whereas the relative abundance of amino acids increases at later stages [40,47,52,53,54]. For instance, young Chinese fir trees exude higher levels of carbohydrates and quercetins, supporting nutrient acquisition for rapid growth, while over-mature trees secrete more lipids and salicylic acids (SA), reflecting a shift toward defensive metabolite production [55].

Plant growth rates correlate positively with exudation quantity and microbial load. Fast-growing plants, especially during exponential growth phases, release larger total amounts of sugars, amino acids, and organic acids. In contrast, plants in slower growth stages release relatively higher proportions of these metabolites, even though the overall quantities are lower [54]. The trade-off between the metabolic cost of exudation and the benefits of attracting beneficial microbes tends to become more favorable in older plants, due to larger root systems and more established rhizosphere microbiomes, although the mechanisms underlying this balance remain poorly understood [46,54].

Exudation is also spatially heterogeneous along the root axis. It is particularly pronounced at the root tip, the first region to explore new soil environments and a major site for sensing and responding to environmental stimuli [40]. Root age further influences exudation: older root segments exhibit lower passive efflux, partly due to cell wall thickening and the development of Casparian strips that restrict solute movement [46]. In addition, root length and surface area generally correlate positively with total exudation [56,57,58], although some studies report no consistent link between specific architectural traits and exudate quantity or composition [59,60,61]. These discrepancies suggest that species-specific differences in root morphology and cellular anatomy play a major role, making broad generalizations across taxa difficult. The lack of consistency across studies also points to non-standardized methodological approaches that hinder direct comparisons.

Ultimately, intrinsic plant traits do not act in isolation; they interact closely with environmental and biological factors, producing highly context-dependent exudation patterns [46]. These extrinsic influences are described in detail in Section 4.2.

3. Root Exudates as Drivers of Plant–Soil–Microbe Interactions

3.1. The Rhizosphere as a Dynamic Interface

The rhizosphere, defined as the soil zone directly influenced by root activity. It represents one of the most dynamic and complex microenvironment on Earth, where intricate exchanges occur between root structures, soil, and associated microbial populations [62]. In fact, all living organisms on Earth rely on inter and intraspecific interactions to survive and evolve [63]. As a high-energy microenvironment, rhizosphere supports a much higher density of microorganisms than the surrounding bulk soil, acting as a biochemical “hotspot”, as a great percentage of photosynthetically derived C is released from plant roots as exudates [64,65]. Microorganisms in the rhizosphere, including bacteria, fungi, archaea, and other soil organisms, form complex communities shaped by both the composition of root exudates and soil properties [66].

Among the different models of plant-microbe interactions, symbiotic relationships represent the most significant and evolutionarily relevant form of association [67]. These mutualistic interactions often lead to reciprocal benefits, enhancing plant nutrition, growth, and stress tolerance. However, not all associations are advantageous. Certain microorganisms, including bacteria, fungi, and viruses, have evolved strategies to exploit plant resources, invade host tissues, and ensure their own survival, often causing diseases (i.e., pathogenesis) [67]. In contrast, commensal relationships involve interactions between two distinct species in which one organism benefits while the other remains unaffected, experiencing neither positive nor negative consequences [68].

3.2. Root Exudates as Mediators of Microbial Recruitment and Chemical Communication

At the interface between plant roots and soil, exudates act as primary mediators of ecological and molecular interactions, defining the structure and function of the rhizosphere microbiome. Different compounds selectively attract beneficial microorganisms, while inhibiting pathogens. For instance, amino acids and sugars act as energy sources for copiotrophic bacteria, whereas flavonoids, strigolactones, and terpenoids function as specific signaling molecules triggering symbiotic associations [69,70]. Previous studies have suggested that plants actively select specific subsets of microbes at distinct developmental stages, likely to fulfill specialized functional roles [69]. This selective recruitment is increasingly viewed as part of a plant “cry for help” strategy, whereby stressed plants modulate root exudation patterns to attract beneficial microbes that enhance protection, nutrient acquisition, and overall resilience [71]. These compounds mediate key biological processes such as nodulation [72,73], mycorrhizal symbiosis, the establishment of plant growth-promoting rhizobacterial (PGPR) consortia, and endophytic associations. They also contribute to antimicrobial activity, inhibition of biofilm formation, quorum-sensing interference, and tri-trophic interactions involving nematodes [74,75].

AMF form symbiotic associations with the roots of over 80% of terrestrial plant species [66], improving P uptake, as these fungi extend the root system’s absorptive surface area, enabling better access to phosphate ions that are otherwise immobile in the soil [76]. Under P limitation, roots release strigolactones (SLs) that stimulate spore germination and hyphal branching in AMF [66], leading to the growth and metabolic activation of AMF and facilitating host recognition and colonization [77,78,79]. SLs also induce the release of chitin oligomers by AMF, reinforcing plant–fungus communication [80,81]. Additionally, the GlcNAc transporter NOPE1 has been implicated in early symbiotic signaling, suggesting the existence of GlcNAc-based compounds that trigger presymbiotic fungal responses [70,82]. In parallel, AMF secrete GlcNAc-containing molecules, collectively termed Myc Factors, including lipo-chitooligosaccharides (Myc-LCOs) and short-chain chitin oligomers (Myc-COs), which elicit calcium spiking and activate symbiotic gene expression in the host [81,83]. This reciprocal exchange of diffusible signals highlights the finely tuned bidirectional communication that orchestrates the establishment of plant-AMF symbiosis [70].

Similarly, isoflavonoids exuded by leguminous plants, such as genistein and luteolin [66], serve as signaling compounds that activate Rhizobium species, leading to the formation of N-fixing nodules, thus exemplifying another strategy by which plants recruit and sustain beneficial symbionts in the rhizosphere [67]. These flavonoids induce the expression of nod genes in Rhizobium, resulting in the synthesis of lipo-chitooligosaccharide Nod Factors, which are recognized by specific plant receptors to initiate nodule organogenesis and infection thread formation [73,84]. This symbiotic relationship greatly increases N availability in the soil, thereby reducing dependence on synthetic nitrogen fertilizers [85]. The high specificity of the flavonoid–Nod factor signaling pathway enables legumes to selectively associate with compatible Rhizobium strains, maximizing N fixation efficiency. For instance, soybean roots grown on agar release specific flavonoids that attract Bradyrhizobium japonicum, a highly effective N-fixing bacterium. Such selective recruitment has important implications for agriculture, as it enhances soil fertility while minimizing the environmental impact associated with N fertilizer use [66].

In addition to symbiotic fungi and rhizobia, PGPR in general are also selectively recruited by root exudates (Table 1) through nutrient-mediated chemotaxis and receptor-based signaling.

Table 1. Representative examples of root exudate chemoattractants and their associated plant growth-promoting rhizobacteria.

Plant Species	Root Exudates	PGPR Recruited	Effect
Arabidopsis thaliana [86]	L-malic acid	Bacillus subtilis FB17	Enhances root colonization, strengthens induced systemic resistance, and promotes plant growth.
Citrullus lanatus [87]	Malic and citric acids	Paenibacillus polymyxa	Enhances nutrient acquisition, promotes root and shoot growth, improves stress tolerance, and provides biocontrol against soil-borne pathogens.
Zea mays [88]	Tricarboxylic acid	Pseudomonas putida	Enhances nutrient uptake, promotes root architecture remodeling, induces systemic resistance, and increases tolerance to abiotic stress.
Leguminous species [20]	Amino acids (glutamine, proline) and monosaccharides (glucose, frutose)	Rhizobium spp. Streptomyces spp.	Enhances pathogen resistance, promotes nutrient cycling and improves symbiotic N fixation

These bacteria, which include species of Pseudomonas, Bacillus, and Azospirillum, respond to gradients of amino acids, sugars, and organic acids in the rhizosphere that serve as both chemoattractants and C sources, facilitating their movement and adhesion to root surfaces [20,89]. Under controlled conditions, organic acids act as chemoattractants, facilitating the colonization of PGPR like Bacillus velezensis B26 (Bacillales: Bacillaceae) and B. amyloliquefaciens SQR9 (Bacillales: Bacillaceae), protecting plants from soil-borne pathogens [23]. Likewise, amino acids function as signaling molecules that attract and enrich beneficial rhizosphere microorganisms [21]. The perception of these compounds is mediated by methyl-accepting chemotaxis proteins (MCPs), transmembrane receptors that recognize ligands such as malic, citric, and fumaric acids, amino acids (e.g., alanine, glycine, or glutamine), and sugars (e.g., ribose, maltose). Ligand binding activates the CheA–CheY phosphorylation cascade, resulting in flagellar rotation and directional movement toward the rhizoplane [20,90].

Representative studies, under controlled conditions, show the attraction of Bacillus subtilis FB17 by L-malic acid exuded from Arabidopsis thaliana, which enhances chemotaxis, biofilm formation, and induced systemic resistance [86]; recruitment of Paenibacillus polymyxa by malic and citric acids in watermelon (Citrullus lanatus) [87]; and the stimulation of Pseudomonas putida colonization in maize by tricarboxylic acid intermediates [88]. Compounds such as glutamine and proline recruit microorganisms including Streptomyces and Rhizobium, enhancing pathogen resistance and nutrient cycling [20]. Moreover, compounds such as proline, organic acids, and hormones are exuded in response to water and salt stress, enabling the selective recruitment of beneficial microorganisms [91,92]. Under drought conditions in controlled experiments, plants preferentially associate with Bacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, Azorhizobium caulinodans, and Rhizobium leguminosarum [93]. Whereas salinity stress under in vitro conditions favors the recruitment of Bradyrhizobium japonicum, Mesorhizobium ciceri, and PGPRs such as Pseudomonas fluorescens, Pseudomonas psychrotolerans, Bacillus amyloliquefaciens, and Bacillus subtilis [94]. Together, these microbial assemblages enhance nutrient availability, root colonization, and stress tolerance, contributing to improved plant resilience under adverse conditions [93,94].

Monosaccharides like glucose and fructose provide accessible C sources that support these microbial communities and strengthen plant–microbe symbioses [20].

The specificity of this recruitment is mediated by the activation of bacterial chemotaxis and adhesion genes, which regulate flagellar motility, biofilm development, and surface attachment. Once recruited to the rhizoplane, PGPR colonization depends on the formation of biofilms stabilized by extracellular polymeric substances (EPSs) composed of polysaccharides, proteins, and lipids. Root exudates containing compounds such as gluconic acid and proline enhance this process, promoting durable adhesion and conferring resistance to desiccation and pathogen invasion [20,95]. Mutant strains lacking specific chemotaxis or adhesion genes (cheA, cheY, lapA, lapF) display reduced colonization efficiency, demonstrating the genetic regulation underlying these interactions [96]. This recruitment and biofilm development are coordinated through quorum-sensing (QS) systems based on acyl-homoserine lactones (AHLs) and small peptides that enable PGPR populations to synchronize motility, metabolism, and biofilm maturation. Interestingly, certain root exudates mimic AHL-like molecules, allowing plants to interfere with bacterial QS and selectively promote beneficial taxa [89,97].

Altogether, root exudates act not only as a “carbon currency” sustaining rhizosphere microorganisms but also as “molecular languages” that guide the recruitment, attachment, and cooperative behaviour of microbial communities, forming the biochemical foundation of plant-microbe mutualism [20].

3.3. Microbial Feedbacks and Adaptive Regulation of Plant Performance and Root Exudation

The influence of root exudates is not unidirectional (Figure 1). Microbial colonization, enzyme secretion, and nutrient turnover also reshape exudate profiles (quantity and composition), creating feedback mechanisms that further modulate plant adaptation. Increasing evidence shows that beneficial rhizosphere microorganisms produce signaling molecules capable of modulating root system architecture and shoot development [63], ultimately reshaping the biosynthesis of metabolites released into the rhizosphere [98]. Indeed, microorganisms secrete metabolites such as lipo-chitooligosaccharides, volatile organic compounds (VOCs), and siderophores that act as regulatory feedback cues, regulating root architecture, secondary metabolism, and defense activation. Stress-induced exudation can further modulate hormonal signaling cascades linked to abscisic acid (ABA) and SA, strengthening systemic tolerance [99].

Figure 1. Representation of the bidirectional interactions between root exudates and soil microorganisms, supported by evidence from multiple studies discussed in Section 3 of this review. Abbreviations: AHL—acyl-homoserine lactones; AMF—arbuscular mycorrhizal fungi; CO—chitin oligomers; EPS—extracellular polymeric substances; LCO—lipo-chitooligosaccharids; PGPR—plant growth-promoting rhizobacteria.

Beyond these local rhizosphere interactions, microbial VOCs also function as airborne regulators capable of triggering long-distance plant responses. Compounds such as 2,3-butanediol, acetoin, and terpenoid derivatives can upregulate photosynthesis-related genes, enhance stomatal conductance, and stimulate biomass accumulation even without physical root contact [100,101,102]. In addition, microbial production of auxins and cytokinins, as well as the regulation of ethylene levels, plays a decisive role in shaping plant morphology, stress regulation, and overall growth performance [103].

Inoculation with PGPR profoundly influences rhizosphere chemistry by modulating both the quantity and biochemical profile of root exudates. Numerous studies have shown that strains such as Pseudomonas fluorescens and Bacillus subtilis can upregulate the secretion of organic acids, amino acids and phenolic compounds, thereby enhancing nutrient solubilization, metal chelation and the recruitment of beneficial microorganisms [104]. Organic acids like malate, citrate and oxalate also act as chemoattractants that shape microbial assembly patterns in the rhizosphere [105,106]. Phenolics released under PGPR influence similarly contribute to microbial selection and the suppression of soil-borne pathogens, reinforcing both nutrient cycling and plant defense [107].

During AMF symbiosis, fungal colonization strongly alters plant C flows by diverting a substantial share of root-derived sugars and lipids toward the fungal partner. This increased belowground C allocation stimulates the production and release of diverse root exudates, which support fungal development, enhance nutrient mobilization and modulate rhizosphere interactions [108,109].

Taken together, root exudation is a key interface in plant–microbe feedback loops. Rather than acting as static chemical inputs, root exudates participate in dynamic, multi-level communication networks, whose effects emerge from the integration of microbial responses and plant physiological status. Many of the studies cited have reported associations between specific exudates, particular microbial groups, and individual plant or soil traits, typically examined in isolation. However, under natural conditions, multiple exudates, diverse microbial communities, and interconnected plant and soil factors operate simultaneously, and the resulting interactions can differ substantially from those observed in simplified systems. Moreover, most insights reported in the literature are derived from experiments conducted under controlled conditions, which may not capture the full complexity of field environments, emphasizing the need to bridge laboratory and field studies. Developing a deeper understanding of these feedback-driven interactions is therefore essential for accurately interpreting plant–microbe dynamics.

4. Multifunctional Roles of Root Exudates in Soil Ecosystems, Stress Adaptation and Plant Interactions

4.1. Influence of Root Exudates on Carbon Dynamics and Overall Soil Health

Carbon fixed from atmospheric CO₂ through photosynthesis enters the soil via aboveground plant residues, root exudates, and root turnover, which together represent the primary sources of soil organic carbon (SOC) [27,110]. In particular, root exudation represents a substantial C investment for plants, with estimates suggesting that 5–21% of net photosynthetically fixed C is allocated to the rhizosphere through this process [111,112,113]. Thus, these metabolites play a central role in the soil C cycle, serving as dynamic mediators between plant photosynthetic activity and SOC processes. However, there is a paradox in the literature concerning the contribution of root exudation to the stabilization and destabilization of SOC [110]. SOC is a dynamic organic continuum that was classified by Lavallee et al. (2020) into particulate organic C (POC) and mineral-associated organic C (MAOC) [114,115]. While the POC fraction is more vulnerable to microbial decomposition, the MAOC displays higher persistence due to protection by mineral association [116].

One of the most important mechanisms through which exudates interfere with the soil C cycle and the decomposition of stable SOC is the so-called priming effect [115]. The readily available C compounds in root exudates serve as an immediate energy source for soil microorganisms, thereby stimulating SOC decomposition by altering the structure, composition, and activity of microbial communities. This microbial stimulation often leads to the accelerated decomposition of existing soil organic matter (SOM), thereby releasing additional CO₂ to the atmosphere, a phenomenon known as a positive priming effect [115]. Other hypothesis posits that specific root exudates, such as oxalic acid, can destabilize MAOC complexes, leading to the release of previously protected C fractions and enhancing their availability for microbial decomposition and utilization. However, under certain environmental or nutritional conditions, exudates can also induce a negative priming effect, where microbial activity promotes stabilization, by promoting the formation of stable MAOC and facilitating aggregate formation that protects carbon from rapid decomposition [110,117,118].

On the other hand, microbial activity promoted by root exudates, not only enhances decomposition but also contributes to the formation of new, stable organic matter. As microbial communities proliferate and undergo turnover, a portion of their biomass is converted into microbial residues, which can become strongly associated with mineral surfaces or physically protected within soil aggregates [119,120]. These microbially derived C pools exhibit enhanced physicochemical stability, constituting a major component of long-term C sequestration. Consequently, although root exudates primarily supply labile C that is rapidly metabolized, they indirectly promote the accumulation of stable SOM through microbial transformation and stabilization processes [119].

This duality of root exudates effect on C dynamics underscores their complex role, acting either as drivers of C mineralization or as agents of stabilization, depending on several mechanisms and conditions [115]. These include soil physicochemical properties, plant species, microbial composition, chemical composition of released compounds, nutrient availability, climate, and already existing C pools in the soil [110,117,121]. In particular, the composition of exudates plays a crucial role in modulating soil C dynamics, as exudates with simpler C structures are more easily metabolized by soil microorganisms, resulting in faster energy conversion and a stronger priming effect. In contrast, compounds with more complex molecular structures tend to elicit a weaker priming response [122]. Furthermore, an increase in the rate of root exudation generally intensifies the priming effect, thereby enhancing the decomposition of SOM [115].

Despite progress in understanding root exudate-microbe interactions, the magnitude, direction, and persistence of their effects on soil C cycling remain uncertain, and the mechanisms behind the rhizosphere priming effect are still poorly understood [115].

Beyond their central role in C cycling, root exudates are key drivers of overall soil health, influencing the biological, chemical, and physical properties of the rhizosphere [123]. By stimulating microbial growth, these metabolites promote the formation and stabilization of soil aggregates, as microorganisms physically bind soil particles through fungal hyphae, produce adhesive biofilms, and release EPS and glomalin that enhance cohesion between mineral and organic fractions [124]. As a result, well-structured aggregates enhance soil porosity, water infiltration, and water-holding capacity, while protecting organic matter from rapid decomposition and reducing susceptibility to erosion [125,126]. Moreover, root exudates facilitate nutrient mobilization by shifting rhizosphere pH and chelating metal ions, and contribute to the suppression of soil-borne pathogens, thereby improving the plant’s overall tolerance to stress [127,128]. These mechanisms are discussed in deep detail in Section 4.2 of this Review.

4.2. Root Exudates in Plant Tolerance to Abiotic and Biotic Stresses and Their Role in Agroecosystem Resilience

Considering that plants are sessile organisms that cannot escape to unfavorable conditions, they rely on a diverse range of adaptive strategies, including immune responses, dynamic shoot growth, root system plasticity, strategic resource allocation, and the release of allelochemicals and VOCs, to withstand and respond to biotic and abiotic stresses [129,130].

Root exudates play a pivotal role in sustaining plant health and enhancing stress tolerance, acting as key drivers of the rhizosphere priming effect and mediating complex interactions among plants, soil, and microorganisms [131]. As previously mentioned, under abiotic and biotic stresses, plants strategically release these compounds, shaping the composition and activity of the rhizosphere microbiome, and promoting ecological feedback to plant hosts [131,132,133]. The composition and rate of root exudation are strongly influenced by the type and intensity of stress, which trigger distinct physiological and metabolic adjustments (Table 2) [134].

Table 2. Summary of major abiotic and biotic stresses, associated changes in root exudate profiles, and the primary functions of these exudates in supporting plant stress tolerance.

Type of Stress	Root Exudate Profile	Function	References
Drought	Amino acids; sugars; organic acids	Osmotic adjustment; protection of cellular structures	[7,135,136]
Salinity	Amino acids; fatty acids; phenolics	Osmotic balance; stabilization of membranes	[94,137]
Temperature	C-rich compounds	Metabolic activity; nutrient uptake via stimulated microbial activity and nutrient cycling	[138,139]
Nutrient deficiency	Carboxylates; phosphatases; amino acids	Mobilization of soil nutrients; nutrient uptake	[11,109]
pH imbalance	Carboxylates; protons (H+)	Rhizosphere pH regulation; improved nutrient solubility	[5,109]
Pathogen infection and herbivore attack	Phytohormones; VOCs; phenolics	Activation of defense signaling; antimicrobial activity; signaling to neighboring plants	[140,141]

Under drought and salinity stress, plants often increase the exudation of osmoprotectants that facilitate osmotic adjustment, enhance the solubilization and mobilization of soil minerals essential for nutrient uptake, and release signaling molecules that promote associations with stress-tolerant microbial communities [7,135,136]. Among these compounds, the amino acid proline, an important osmotic regulator that protects plant cells, by stabilizing proteins and membranes and maintaining cellular water balance, has been reported to accumulate in the root exudates of several plant species under drought conditions [91,92,133]. However, additional compounds are also released in response to drought. In cotton (Gossypium hirsutum) grown under growth chamber conditions, drought stress has been shown to enhance root exudation of organic C, organic N, and free amino acid-N. These exudates play critical roles in rhizosphere biogeochemical cycling, reflecting the tight coupling of C and N dynamics mediated by microbial communities [142]. Similarly, in a greenhouse experiment, blue grama (Bouteloua gracilis) exhibited increased exudation of TOC and organic C under drought stress [143]. On the other side, under controlled conditions, rice plants (Oryza sativa L.) exposed to drought stress showed increased exudation of ABA [144].

As with drought conditions, Citrus macrophylla plants grown in vitro and subjected to salt stress also release high amounts of proline into the rhizosphere [92,94]. In Phragmites australis exposed to short-term salt stress and grown under natural light and uncontrolled temperature conditions, root exudates contained a broad spectrum of amino acids, with proline and histidine being the most abundant. In addition, several fatty acids and benzene derivatives, including palmitic acid, 2-methylbutyric acid, stearic acid, palmitoleic acid, 1,3,5-tris(1-methylethyl)-benzene, and eugenol, have been reported as key classes of compounds exuded by plants growing in saline soils [94,137].

In turn, under nutrient-limited conditions, plants adjust their root exudation profiles by increasing the release of carboxylates, phosphatases, amino acids, and several signalling metabolites, which act to mobilize inaccessible nutrient forms and regulate rhizosphere processes [109]. Phosphorus deficiency is one of the most extensively described in the literature, with numerous plant species showing enhanced exudation of low-molecular-weight carboxylates, including malate, citrate, and oxalate. This strategy is particularly effective for increasing P availability because these carboxylates can mobilize otherwise inaccessible P by displacing it from inorganic and organic soil complexes [11]. The efficiency of P desorption largely depends on both the concentration and chemical structure of the exuded compounds: tricarboxylates (e.g., citrate, aconitate) exhibit the highest capacity to solubilize soil-bound P, whereas monocarboxylates (e.g., acetate, pyruvate) are considerably less effective [11]. In addition, according to Carvalhais et al. (2011), P deficiency induces maize (Zea mays) roots grown under sterile conditions to release higher amounts of γ-aminobutyric acid (GABA), which plays a regulatory role by modulating anion channels involved in malate efflux [145,146]. Malate is a key compound exuded by soybean (Glycine max) to mobilize inorganic P from soil particles [133,147]. In turn, in a field experiment, pigeon pea (Cajanus cajan L.) roots under P deficiency specifically release piscidic acid (p-hydroxyphenyl tartaric acid) [5]. Deficiencies of other macro- and micronutrients also trigger distinct root exudation responses that help plants mobilize otherwise inaccessible nutrient pools. In graminaceous species, Fe deficiency strongly stimulates the release of phytosiderophores compounds (e.g., mugineic acid) that chelate Fe³⁺ in the rhizosphere and greatly enhance its solubility and uptake [148]. Interestingly, similar increases in phytosiderophore release have been reported under Zn deficiency and, to a lesser extent, under Mn and Cu deficiency, although the magnitude of this response varies widely among species and cultivars [149]. Moreover, organic acids, such as citrate and malate, play a central role in mobilizing micronutrients (Mn, Zn, Cu) by acidifying the rhizosphere and forming soluble complexes that facilitate uptake. Phenolic compounds also contribute to the mobilization of redox-active elements, particularly Mn, through their capacity to reduce Mn³⁺/Mn⁴⁺ to a more plant-available form (Mn²⁺). Some grasses additionally exude hydroxamic acids, secondary metabolites that can chelate metals and, in certain cases, act as allelopathic agents that suppress the growth of neighboring plants. Together, these nutrient-specific exudation strategies highlight the versatility of root chemical secretion as a mechanism for coping with nutrient scarcity and restructuring the rhizosphere environment to enhance nutrient acquisition [5].

On the other hand, when plants detect soil pH imbalances, they adjust their exudation patterns to stabilize rhizosphere conditions and maintain cellular homeostasis. For instance, in acidic soils or under conditions of excessive ammonium (NH₄⁺) supply, the preferential uptake of cations over anions leads to proton (H⁺) extrusion by the roots, accompanied by enhanced synthesis of carboxylic acids for internal pH regulation. However, a substantial portion of these carboxylates is retained within the tissue, as organic acid anions are required for NH₄⁺ assimilation, which simultaneously generates additional protons and triggers decarboxylation processes [5]. As a result, both the internal concentrations and exudation rates of carboxylates tend to decline with increasing NH₄⁺ availability. In contrast, in alkaline soils or when nitrate (NO₃⁻) is the dominant N source, plants often release greater amounts of carboxylates and protons into the rhizosphere to acidify the surrounding soil and enhance the solubility of otherwise inaccessible nutrients such as P and micronutrients.

Furthermore, temperature acts as an important abiotic driver of root exudation, often interacting with other environmental stresses. It can act directly by influencing plant metabolic activity and indirectly by modifying soil biological, physical, and chemical properties [46]. Across diverse plant groups, including trees, shrubs, crops, and sedges, root exudation rates, typically measured as total C released, generally increase with rising temperature [138,139,147]. However, the response largely depends on the warming timescale: short-term, acute soil warming (+10 °C) has been shown to immediately stimulate exudation, whereas long-term warming (+4 °C) increases fine-root carbon release primarily through greater fine-root biomass, rather than through higher exudation per unit root mass [150]. Temperature often interacts with other environmental drivers. For instance, elevated temperatures have been shown to increase C exudation when combined with high CO₂ levels, as elevated CO₂ enhances photosynthesis and C supply while warming accelerates plant metabolism, microbial activity, and nutrient cycling, together intensifying plant–microbe C exchange [151,152]. In some systems, elevated temperature stimulated exudation only when N was supplied, suggesting that warming increases plant N demand, and when it is abundant, this promotes greater root growth and exudation [10].

Plants are continuously challenged by pathogenic microorganisms and pest insects, and their survival depends on the ability to accurately recognize these threats and activate defense mechanisms to counteract and suppress pathogen attack [153]. As a first line of defense, plants rely on pattern recognition receptors (PRRs) located on the cell surface, which detect highly conserved pathogen-associated molecular patterns (PAMPs), such as flagella, membrane lipopolysaccharides (LPS) and other cell wall or secreted components [131,154]. Non-pathogenic microorganisms are perceived in a similar manner, and their elicitors are termed microbe-associated molecular patterns (MAMPs) [155]. In addition to flagella and LPS, plants can detect several other bacterial metabolites, such as N-acyl-l-homoserine lactones, biosurfactants, siderophores, and the antibiotics DAPG and pyocyanin [131,154]. Recognition of these PAMPs/MAMPs triggers a cascade of defense responses that reinforce plant resistance to infection [155].

Upon recognition of biotic stresses, plants activate systemic acquired resistance (SAR) and induced systemic resistance (ISR), two signaling pathways that establish a primed defensive state through the activation of a suite of defense-related genes and signaling proteins [140,156]. These systemic responses are closely linked to changes in root exudation. For instance, under controlled conditions, Pseudomonas syringae infection in Arabidopsis thaliana modifies the expression of root malate transporters, leading to increased malic acid secretion and the subsequent recruitment of protective Bacillus subtilis populations to the rhizosphere [140,141]. Similarly, under field conditions, cucumber plants infected with Fusarium oxysporum f. sp. cucumerinum enhance the exudation of fumaric and citric acids, which promote Bacillus amyloliquefaciens biofilm formation and strengthen plant–microbe interactions involved in disease suppression [140,157].

In addition, when plants are attacked by multiple herbivores, they can differentially engage SA and jasmonic acid (JA) signaling pathways. Research has shown that the interaction between SA and JA pathways can be either synergistic or antagonistic, depending on the context [158,159]. Moreover, herbivores release specific salivary effectors during feeding, which can directly or indirectly modulate the activation of these hormonal pathways [160]. For instance, under controlled conditions, potato plants infested by necrotrophic herbivores primarily activate JA-dependent responses, whereas attacks by biotrophic insects predominantly trigger SA-mediated signaling [161,162]. Overall, root exudation represents one of the most dynamic plant adaptive responses to environmental constraints, enabling plants to actively modulate their immediate surroundings and sustain functionality under stress. This process functions as a central regulatory mechanism governing plant–soil–microbe interactions through dynamic chemical signaling and resource exchange. Accordingly, Figure 2 provides a synthetic representation of the plant–soil–microbe interactions discussed throughout this review, summarizing the collective insights derived from the cited literature.

Figure 2. Evidence-based graphical summary of plant–soil–microbe interactions addressed in this review. The scheme integrates findings from multiple experimental studies cited throughout the manuscript, illustrating how root exudates mediate microbial recruitment, soil processes, and plant functional responses. Abbreviations: ABA—Abscisic acid; AMF—Arbuscular mycorrhizal fungi; GABA—Gamma-aminobutyric acid; JA—Jasmonic acid; PGPR—Plant growth-promoting rhizobacteria; SA—Salicylic acid.

Although the reported studies offer valuable insights into the exudation of specific compound classes under individual stresses, such patterns do not always translate directly to field conditions, where multiple biotic and abiotic factors interact simultaneously. This complexity highlights the need for integrative approaches, moving beyond isolated interactions to capture the full spectrum of root exudate dynamics in climate change scenarios, where plants frequently face multiple, overlapping stresses.

As abiotic and biotic stresses are expected to intensify under climate change, including more frequent heatwaves, irregular precipitation, and altered pest and pathogen pressures [3], the capacity of plants to adjust rhizosphere processes becomes increasingly critical. In this context, root exudate-mediated responses contribute to agroecosystem resilience by regulating soil C and nutrient cycling, shaping beneficial microbial communities, and enhancing plant defense mechanisms. At the same time, these responses may involve trade-offs, including increased C allocation to exudation at the expense of plant growth and stimulation of microbial activity that can accelerate SOM turnover. Nevertheless, by sustaining soil functionality and plant performance under stress, root exudation remains a key mechanism supporting plant and soil health and promoting agroecosystem stability under climate-driven pressures [163] (Figure 3).

Figure 3. Conceptual schematic representation of root exudates as dynamic mediators of plant adaptation to environmental stresses and contributors to agroecosystem resilience, under a climate change scenario.

4.3. Recognition of Neighboring Plants

Under limited or stressful conditions, plants show remarkable plasticity, adopting competitive strategies to secure resources or forming mutualistic, co-evolutionary associations that improve their chances of survival [131]. Plant signaling and its associated responses are central to these outcomes, shaping plant performance, coexistence, and overall community structure. The ability of plants to sense their environment and perceive signals from neighboring individuals provides valuable insight into how plant communication and interactions occur within ecosystems [164]. These interactions are largely shaped by neighbor recognition mechanisms mediated through chemical signaling networks, particularly root exudates and VOCs, which allow plants to discriminate between intraspecific and interspecific neighbors and optimize resource allocation [165]. Recognition relies on specific receptors, including ABC transporters, which perceive these chemical cues and activate downstream physiological and morphological responses [165,166,167]. Once recognition occurs, a cascade of responses unfolds across multiple biological levels. At the individual level, plants may adjust their growth patterns, such as by altering morphology to avoid shade or modifying reproductive structures. Concurrently, they may activate physiological defense pathways, including increased production of secondary metabolites. Over time, these individual-level adjustments can translate into population-level adaptations, promoting niche specialization and ultimately enhancing overall fitness [167].

Interspecific recognition is predominantly associated with allelopathy, a defensive or competitive strategy in which plants release allelochemicals, such as phenolic compounds, terpenoids, and various alkaloids, into the rhizosphere. Such allelopathic signaling is especially prominent in interactions with invasive weed species, often inhibiting the germination, growth or competitive ability of neighboring plants [131,167,168]. Allelopathy thus provides a competitive advantage, allowing plants to secure critical resources such as water, nutrients, and space [164,169,170]. This mechanism is especially well documented in interactions involving invasive species, which often rely on strong allelopathic interference to dominate native communities, as well as in relationships with parasitic plants, where chemical cues are essential for host detection. A study demonstrated that Pisum sativum roots undergo marked spatial adjustments when exposed to Festuca glauca rhizosphere signals, including changes in primary root angles and a redistribution of lateral root biomass [167]. Desmodium spp., in turn, was described to release potent C-glycosyl flavonoids that interfere with the germination and early establishment of Striga hermonthica, effectively suppressing this parasitic weed and reducing its ability to attach to host roots [171]. Several studies have demonstrated that Sorghum bicolor L. releases phenolic acids, such as protocatechuic, p-hydroxybenzoic, syringic, sinapic, p-coumaric, and benzoic acids, through its root exudates, which can function as natural bioherbicides by suppressing the germination and growth of competing weed species [172,173]. Moreover, Kong et al. (2018) reported that (–)-loliolide and JA play key roles in belowground neighbor detection and allelochemical signaling in wheat, suggesting that these compounds may act as widespread mediators of root-mediated plant–plant interactions [169].

In contrast, intraspecific recognition takes place when plants detect conspecific neighbors, including genetically related and unrelated individuals. Unlike interspecific interactions, these processes are driven not by toxic or inhibitory compounds but by informational signaling molecules present in root exudates, such as amino acids, small peptides, hormone-like substances, and non-toxic secondary metabolites [164]. These cues allow plants to assess kinship and modify their behavior accordingly. Many species show forms of kin recognition, where plants reduce competitive intensity when growing alongside genetically related individuals. This can manifest as reduced lateral root proliferation, less aggressive nutrient foraging, or altered biomass allocation patterns [174]. The first evidence of kin recognition in plants was reported in the American searocket (Cakile edentula). This study showed that when growing beside relatives, searocket plants restricted their root growth, reducing competition and allowing shared access to resources. In contrast, when grown next to unrelated individuals, they expanded their root systems, increasing belowground competition [174]. A similar behavior was observed in rice, where plants grown alongside unrelated cultivars developed more extensive and competitive root systems than those grown with closely related lines [175]. In another study, common bean plants adjusted their root system when sensing a neighboring kin plant, reducing root growth in soil zones already occupied by the neighbor and reallocating roots both vertically and horizontally [176]. Root exudates play an essential role in this plant neighbor recognition, as demonstrated by several studies. In Deschampsia cespitosa, roots exposed to exudates from non-sibling conspecifics developed more extensive branching and increased root length compared with those receiving exudates from siblings [177]. Comparable results were found in Arabidopsis thaliana, where plants treated with exudates from unrelated individuals formed a greater number of lateral roots than those exposed to kin-derived exudates. Notably, when exudate secretion was experimentally suppressed, these differences disappeared, confirming that chemical cues within root exudates drive these kin-specific growth responses [178]. This ability to differentiate between kin and non-kin may allow plants to optimize competitive strategies and adjust their growth in ways that improve survival under changing environmental conditions [174].

Figure 4 provides an overview of the ecosystem services promoted by root exudates, highlighting their contribution to enhancing plant tolerance to both abiotic and biotic stresses.

Figure 4. Conceptual illustration of the functional contributions of root exudates to soil–plant interactions, microbial activity, and plant stress adaptation. Abbreviations: AMF—arbuscular mycorrhizal fungi; PGPR—plant growth-promoting rhizobacteria; SOC—soil organic carbon.

5. Methodological Advances in Studying Root Exudates

Deciphering the role of root exudates is complicated, not only due to their low concentration and chemical diversity but also because of their rapid modification in the rhizosphere [179,180]. In fact, root exudates can be one of the most complicated metabolites to assess and quantify, due to the complexity of the soil matrix and their interactions with the surrounding medium [181,182]. In natural systems, separating plant-derived metabolites from those originating from microbes or soil processes is inherently difficult, and exudation itself is modulated by diverse biotic and abiotic cues whose specific contributions remain insufficiently understood. Moreover, the compounds ultimately detected in exudates are strongly dependent on the experimental setup and analytical approach employed [181]. Capturing undisturbed exudates is challenging, as these metabolites can undergo immediate alterations upon release, driven by diverse soil processes such as microbial decomposition, adsorption to the soil matrix, as well as mass flow and diffusion, all of which rapidly change the quality and quantity of the sample [6,99,128,183,184]. Additionally, the plant exudation profile is dynamic and actively influenced by the microbiome, with freshly released compounds being inevitably mixed with dissolved organic C from soil and microbial metabolites [40,99,179,183]. At the same time, the physical inaccessibility of roots poses a major logistical challenge, since collecting samples without disturbing or damaging the plant is difficult [185]. Nonetheless, even with these constraints, traditional methods have long been employed to collect root exudates, attempting to capture these metabolites with as little disturbance or alteration to the root environment as possible [181,186]. Methods relying on inert substrates or simplified soil-free systems have yielded valuable foundational knowledge, yet their capacity to represent the natural complexity of plant–soil–microbe interactions is inherently limited. Despite their limitations, these techniques have provided the baseline upon which more recent methodological advances have been developed.

5.1. Advances in Exudate Collection: From Ex Situ to In Situ Sampling

The limitations inherent to conventional exudate collection methods have prompted substantial methodological innovation over the past years, with new approaches aiming to capture root-derived compounds under conditions that more closely approximate the natural rhizosphere [181,186]. The central challenge remains the same, especially in in situ conditions: to recover exudates with minimal disturbance to the plant and its associated microbiome, while preserving their native chemical composition and spatial–temporal dynamics. Furthermore, different sampling strategies do not merely differ in technical implementation but fundamentally constrain the type of ecological and functional questions that can be addressed, calling for an integrative framework to assess the complexity of root exudate research [187].

The most common exudate collection strategies often rely on artificial experimental setups based on inert substrates, with sampling being performed through destructive procedures such as whole-root harvesting, sectioning, rinsing, or substrate removal. These interventions disrupt root integrity and profoundly alter exudation patterns [186]. Although such methods have been instrumental for establishing foundational knowledge on exudates, they inevitably influence the results, as plants are removed from natural soil conditions, where environmental factors such as chemical gradients and microbial processes are tightly intertwined with root physiology [99,188]. Therefore, substantial effort has been directed toward improving sampling methods, such as hydroponic, semi-hydroponic, soil-based, and soil–water hybrid approaches, in an effort to better reproduce undisturbed root–soil interactions while increasing the reliability of exudate profiles obtained [13,179,181,189]. For instance, hydroponic systems, along with semi-hydroponic systems, are widely used due to their sterility and analytical convenience, but do not fully represent the normal rhizosphere conditions and may induce mild stress responses that alter root growth and exudation patterns [13,186,190]. In fact, these growth conditions actually alter root architecture and rhizosphere organization due to their unique factors, inducing anatomical and physiological changes that modify exudation patterns and limit the extent to which the resulting data reflect plant conditions in natural environments [191].

On the other hand, soil-based approaches and soil–water hybrid systems can help maintain greater realism, while allowing the evaluation of (a)biotic stress, primarily drought [181,184,192]. Nonetheless, these typically require root washing prior to or during sampling, leading to tissue damage and metabolic perturbation, as well as having limited microbial communities due to the need for sterile or semi-sterile conditions, altering the collected exudates [179,184,193]. Consequently, while soil-based and soil–water hybrid approaches enhance ecological realism, the need for root handling and partial microbial simplification may bias exudate profiles and limit the comparability across studies employing different soil-based designs.

Another promising approach that limits root disturbance involves the use of exudate sorption or diffusion traps, which can be used in rhizoboxes incorporated with a transparent side to visualize and access part of the root system [194,195]. In this setup, plants are grown in soil, root development can be monitored through an observation window, and exudates can be sampled more precisely from defined root regions using “exudation traps” [189,194,196,197]. Because the method induces only limited stress, repeated sampling becomes more feasible, especially considering that exudates are released in very small quantities and within narrow zones around roots, and they can be rapidly assimilated by rhizosphere organisms [40]. However, these approaches capture only a partial representation of rhizosphere processes and are sensitive to experimental design parameters such as trap material and exposure duration. Thus, the use of microdialysis systems offer a promising alternative for achieving finer spatial and temporal resolution of exudation dynamics, but their application in soil matrices can be challenging [195,198,199,200]. Nonetheless, even though hybrid approaches partially control for environmental variation, methodological details, such as the exact setup, the collection medium, duration, additives and the recovery phase for the plants, vary considerably among studies [6,180,186]. Despite their high reproducibility and analytical convenience, these experimental setups are still not representative of the full complexity of the rhizosphere under field conditions, where exudation patterns are shaped by plant age, environmental variability, soil properties, and microbial communities, conditions that differ greatly between experimental works [40,41,181,201,202]. This heterogeneity constitutes a major source of bias when extrapolating controlled-environment findings to agroecosystem functioning, as such systems may overrepresent readily available soluble metabolites and obscure the time-dependent dynamics that occur under realistic conditions [191]. Consequently, while these approaches are well suited for primary, mechanistic, and comparative studies, their capacity to support inference at the agroecosystem scale remains limited, underscoring the need for further development of realistic field-based sampling methodologies.

Field sampling methodologies suffers largely from the same problems as any soil-medium experimental setup: exudates are released in small quantities in narrow zones around roots, being rapidly assimilated by rhizosphere organisms. These constraints result in highly localized and temporally variable signals that are difficult to capture quantitatively, being further intensified by the increased practical difficulty of accessing roots for proper sampling [203]. As a consequence, field-derived exudate data are often sparse and sensitive to sampling timing and spatial resolution, complicating cross-study comparability even more. Nonetheless, efforts are being made to improve knowledge under these conditions, with the most common methodologies consisting of isolating and trapping exudates from intact roots, using different mediums, and sampling at different times for a broader exudate spectrum [181,185,186,203]. In parallel, there has been some development in field sampling systems capable of quantifying exudates under realistic agronomic conditions. These tools are designed to operate within the physical and biological complexity of intact soil, moving beyond the constraints of pot-based experiments. The previously mentioned microdialysis units function by mimicking a root-like probe, where a semi-permeable membrane is perfused with a solution, and compounds diffuse across it based on the soil’s concentration gradient, allowing for continuous, non-destructive sampling of low-molecular-weight metabolites like organic acids and sugars with high spatial resolution [198,204,205]. Perfusion systems, including micro- or normal lysimeters can provide integrative samples over a larger soil volume, though they require careful management to avoid altering soil moisture gradients and potentially inducing artificial exudation responses [206,207].

Collectively, controlled-environment methods and field-deployable advances can complement each other and help bridge the gap between mechanistic laboratory studies and real-world plant–soil interactions. They address the core challenge of capturing processes that are governed by native soil structure, a full microbial community, and fluctuating environmental drivers like temperature and moisture. Nevertheless, most current methodologies remain better suited to identifying potential mechanisms than to quantitatively validating resilience outcomes under agronomic conditions. Consequently, a persistent gap remains between the data obtained in controlled assays and the spatiotemporally complex processes that govern rhizosphere function in the field.

5.2. Challenges in Quantification and Interpretation of the Exudome

Despite the methodological progress of root exudate collection, their proper quantification remains challenging. This complexity arises from the confluence of analytical bottlenecks, the dynamic interplay with soil microbes, the intricate spatiotemporal nature of exudation, and the difficulty of translating chemical data into functional understanding. In fact, these factors do not act independently but interact to systematically constrain both the accuracy and the comparability of exudate profiles across different studies [179].

One of the primary hurdles is the analytical workflow itself, with most root exudate methodologies relying on the identification of these compounds by gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS), with some also using nuclear magnetic resonance (NMR) spectroscopy or Fourier-transform ion cyclotron resonance (FTICR) [187,208]. Techniques like GC-MS and LC-MS are cornerstones for detecting a wide spectrum of metabolites, but present distinct limitations: while GC-MS offers excellent separation and robust spectral libraries, it often requires extensive derivatization; on the other hand, high-resolution LC-MS has broad coverage but suffers from a significant identification gap due to a lack of universal spectral databases and reference standards for plant-specific metabolites [179,182,187,208,209]. NMR spectroscopy is unparalleled for definitive structural elucidation, providing concentration-proportional signals without destruction of the sample but has low sensitivity; while FTICR is cheaper, it only provides the identification of functional groups [208,210,211]. As a result, analytical choices strongly influence which metabolite classes are detected, frequently biasing datasets toward abundant or well-characterized compounds and shaping downstream ecological interpretation. Consequently, even with advanced instrumentation, accurate quantification of root exudates is hindered right at the methodological approach. Furthermore, their low concentrations in the rhizosphere often necessitate sample preparation and concentration prior to analysis, steps that can introduce even more bias, with the chemical background of the growth and collection mediums also being capable of interfering with detection [182,187]. This leads to reported exudate profiles sometimes reflecting methodological accessibility rather than true biological relevance, further reducing cross-study comparability and complicate synthesis across experimental platforms.

Besides these problems, there is also the difficulty in the interpretation of root exudates. For instance, immediate and rapid modifications can happen in the exudome by the rhizosphere microbiome, as these compounds can be metabolized, transformed, or completely consumed by microorganisms even within minutes of being released [106,179,212]. This microbial interference also hinders the cause and effect premise, making it difficult to understand if a detected compound shaped the microbial community or is a product of it [213,214]. This is also connected to another aforementioned interpretation problem, their heterogeneity in both space and time [215,216]. As previously mentioned, exudation is dynamic and happens in response to diurnal cycles, nutrient availability, surrounding microbial communities, or abiotic stress, with most methodologies providing only a snapshot of a moment [40,181,186,202,217]. These temporal limitations restrict the ability to distinguish transient responses from sustained mechanisms underlying resistance, recovery, or adaptive capacity, which are central to agroecosystem resilience [46].

In order to move beyond cataloging of compounds, there is the need to understand their ecological function, be it nutrient mobilization, pathogen defense, or plant–plant signaling. Nonetheless, few exudate studies have performed such validation, calling for the need for increased integrative experimental designs that align sampling strategies, analytical resolution, and ecological endpoints. In conclusion, the path quantification and interpretation of root exudates requires a holistic, integrative framework. It demands careful alignment of sampling methodologies that minimize artifacts with analytical techniques that maximize identification confidence and their functionality.

6. Perspectives and Future Directions

Root exudates represent a promising but still underutilized frontier in plant and soil sciences. Beyond nutrient mobilization and microbial attraction, they sustain soil health, influence biogeochemical cycles, and help buffer crops against climate stress. To translate this into agricultural innovation, future strategies must integrate exudation dynamics into sustainable and climate-resilient farming systems.

Management practices like crop rotations, cover crops, biofertilizers, and organic amendments can stimulate beneficial exudation and shift soil function from chemically driven to biologically regulated processes.

Breeding crops for optimized exudation offers a new dimension of plant improvement. Genetic variation in both the quantity and composition of exudates suggests potential for selecting traits that enhance nutrient acquisition or recruit beneficial microbiota. Advances in genomics, transcriptomics, and metabolomics provide tools to identify genes controlling exudation, enabling marker-assisted selection or genome editing. Complementary approaches in synthetic biology and microbiome engineering may further enhance nutrient cycling and stress tolerance. Integrating exudate dynamics into rhizosphere engineering frameworks, combined with digital soil monitoring, could allow real-time guidance of soil–plant–microbe interactions.

However, significant challenges persist. Accurately quantifying root exudation under field conditions remains difficult due to methodological limitations, high temporal and spatial variability, and the dynamic interactions between roots, soil, and microbial communities. Thus, studies under controlled conditions often focus on isolated stresses or individual plant–soil–microbe interactions, which may not capture the full complexity of natural systems. In real field environments, multiple biotic and abiotic factors act simultaneously, and diverse exudates and microbial networks interact in ways that can lead to outcomes very different from those observed in simplified experiments. Bridging insights from controlled experiments with real ecological contexts is therefore essential and requires integrative approaches that consider multiple concurrent stresses, the diversity of root exudates, and the dynamic interactions within microbial communities. Furthermore, elucidating the molecular and physiological mechanisms that govern exudate composition and release, and linking these processes to ecosystem-scale functions such as nutrient cycling, C sequestration, and soil health, remains a critical gap. Translating exudate knowledge into predictive models and practical applications will require integrated, interdisciplinary efforts across plant physiology, soil ecology, microbiology, and computational systems biology, enabling the development of frameworks to guide resilient, climate-smart agricultural systems.

7. Conclusions

Root exudates are emerging as a cornerstone of plant–soil–microbe interactions with profound implications for agricultural sustainability. They play a pivotal role in helping plants cope with abiotic stresses, such as drought and nutrient limitation, and in defending against biotic threats by recruiting protective microbiota, making them key mediators of crop resilience. Future research should not only elucidate their biochemical complexity but also harness their ecological potential to design next-generation agroecosystems. By linking fundamental knowledge with applied innovation, through breeding, biotechnology, and ecological engineering, root exudates may become key levers for building soils and crops that are productive, regenerative, and resilient to climate change.

The present review provides a holistic and integrative perspective on root exudates, bridging their ecological, physiological, and functional roles with an up-to-date overview of the methodologies used to investigate them, thus offering a broad foundation that supports future advances across multiple research domains.