In response to biotic and abiotic stress, plants produce and release a wide range of biologically active compounds into the rhizosphere, referred to as root exudates (Baetz and Martinoia, 2014). These compounds are involved in a multitude of functions in the ecological interactions with the rhizosphere’s microbial communities. Root exudates not only mediate beneficial plant–microbe interactions but also play a crucial role in defense against pathogens by releasing antimicrobial compounds and chemical cues that shape rhizosphere community structure. In the context of soilborne pathogens such as Aphanomyces euteiches, these exudates can influence infection dynamics by either attracting or repelling motile spores, or by inducing host immune responses (Oburger and Jones, 2018). Due to the complexity of the root system architecture and interactions with the rhizosphere, sampling root exudates in situ while not disturbing the host plant’s natural exudation pattern and surrounding microbiome has been challenging. The lack of suitable non-disruptive sampling has significantly impeded progress in root exudation research.

At present, root exudate metabolomic studies frequently employ discrete sampling methods that necessitate the physical removal of root exudate matrices for sampling (Oburger and Jones, 2018). This approach can alter exudation rates and composition, impacting microbial colonization and activity that influences microbial-mediated processes such as nutrient cycling, pathogen suppression, and overall rhizosphere health (Oburger and Jones, 2018; Escolà Casas and Matamoros, 2021; Wu et al., 2023). Several studies have utilized methods such as exudation traps and rhizoboxes in combination with a root exudate collecting tool (SOIL-REC), which aim to mitigate these disruptions by isolating exudates in a controlled environment and allowing more localized sampling (Shi et al., 2011; Oburger et al., 2013; Vranova et al., 2013). The selective sampling of exudation traps, while useful for targeted studies, is inherently limited as it captures only a subset of the root exudate dynamics (Oburger and Jones, 2018). The use of filter papers and other selective barriers can result in incomplete data, as it may miss critical interactions between different parts of the root system or between the roots and a broader range of soil microorganisms. The act of isolating root segments or employing recirculation systems, as seen in the SOIL-REC method, can induce stress in plant root, which can lead to atypical exudate production. Disturbance in exudation patterns can be particularly problematic due to it altering plant-microbe interaction which, in turn, alters the rhizosphere (Vranova et al., 2013; Oburger and Jones, 2018; Escolà Casas and Matamoros, 2021).

Recently, it has been reported that variables like collection time and diurnal changes affect the composition and abundance of metabolites in root exudates (McLaughlin et al., 2023). These findings underscore the importance of passive sampling, as most metabolites become detectable shortly after exudation begins (McLaughlin et al., 2023). While longer sampling durations may enhance signal strength, they could also mask short-term fluctuations. Therefore, continuous monitoring is essential to capture the complete temporal dynamics of root exudation (Fleishman et al., 2022; McLaughlin et al., 2023). Frequent and/or long period discrete sampling, which involves collecting distinct samples at set intervals, can also lead to a malfunctioning ecological conditions of a continuous system (Vranova et al., 2013; Tian et al., 2021; Fleishman et al., 2022). It was previously shown that there is a high degree of spatial heterogeneity in microbial composition within the rhizosphere, even across a single root (Fleishman et al., 2022). This variation is driven by differences in microbial abundance and diversity relative to the distance from the root surface. Traditional discrete sampling methods often fail to capture this spatial complexity within the rhizosphere, resulting in an incomplete understanding of plant-microbe interactions. Their application in soil environments is limited by challenges such as complex matrices, and the need to physically remove the media, which can disrupt the native rhizosphere conditions.

These challenges necessitate the exploration of approaches that offer ecologically relevant sampling of root exudates in a non-disruptive method. To our knowledge, there is an absence of published studies employing such passive sampling methods for the purpose of root exudate within agricultural research and use in small volume sampling. A coated blade for passive sampling has previously been validated in diverse media, including diluted beef extract, where it effectively sampled veterinary drugs within a short immersion time of 15 minutes (Gómez-Ríos and Pawliszyn, 2014; Gómez-Ríos et al., 2018; Khaled et al., 2020; Kasperkiewicz and Pawliszyn, 2021). Furthermore, passive sampling techniques have also been reported in screening for drugs of abuse in biofluids, pesticides in food, and monitoring of pollutants in aqueous environments (Vrana et al., 2005; Koesukwiwat et al., 2010; Boyacı et al., 2014; Renaud et al., 2022).

The use of passive sampling techniques can be used to monitor and improve our understanding of how specific classes of defense metabolites in plants behave during abiotic and biotic stressors. Isoflavonoids, in particular, are a major class of specialized metabolites in legumes that serve both as signaling molecules in symbiosis and as phytoalexins during pathogen attack. However, how the exudation of these compounds changes during infection, and how they might function as early defense signals, remains poorly understood. This knowledge gap motivated our targeted focus on isoflavonoids as a model class of root exudate metabolites in this study. By leveraging its unique properties, here we employed the coated blade-solid-phase microextraction (CB-SPME) technique to study pea root exudates. In hydroponics, where the root zone is fully immersed in liquid, CB-SPME offers a distinct in situ advantage by passively capturing exuded metabolites without changing the media composition. Its compatibility with both aqueous and complex matrices makes it a versatile tool across systems. CB-SPME serves as both a sample preparation and metabolite extraction method, while traditional methods like solid-phase extraction (SPE) and liquid-liquid extraction (LLE) that are widely used in metabolomics, are best suited for discrete sampling. Furthermore, due to its high sensitivity and increased selectivity based on coating, CB-SPME passive sample collection is superior to conventional passive samplers such as filter paper. Here we present the performance of CB-SPME and compare it with SPE and LLE. To maintain methodological consistency, all methods involved the use of pea root exudates from hydroponic systems, where roots were uniformly exposed to Aphanomyces euteiches zoospores—the causative agent of root rot disease. We report on root exudate metabolite profiles and evaluate the effectiveness of sampling and extraction methods on the relative yield of various compounds, using both a targeted and untargeted metabolomics approach. We also describe the experimental setup and analytical methods used to adopt CB-SPME as a non-invasive, in situ, temporal sampling method for root exudates with the aim to provide a comprehensive resource for researchers interested in adopting passive sampling for similar investigations. This non-destructive sampling technique aligns with the ecological relevance required for studying plant-pathogen interactions.

A predetermined workflow was established for the use of CB-SPME (Restek, Bellefonte, PA) as both the sample collection and metabolite extraction method ( Figure 1 ). Briefly, CB-SPME with HLB-polyacrylonitrile coated blades were preconditioned prior to use. For sampling, blades were directly exposed to the root exudates, allowing in situ adsorption of metabolites onto the sorbent coating. Following exposure, blades were immediately transferred to the LC-MS autosampler vials for desorption with methanol and analyzed directly without additional sample preparation.

Root exudate are traditionally sampled via discrete sampling method where portions of the surrounding media are removed at fixed intervals. This approach disrupts the rhizosphere’s native conditions and may alter metabolite profiles or microbe-root interactions. The challenges of accurately measuring root exudates is also tied to their dynamic nature and rapid turnover rates (Jing et al., 2023; Lei et al., 2023). Therefore, non-invasive and accurate measurement methods for root exudates are essential to preserve rhizosphere integrity and reliability in results. In this study, we assessed the metabolite profile of root exudates using three different extraction methods via discrete sampling, and then employed an in situ root exudate passive sampling technique that removes the disruptive aspects associated with discrete sampling. The extraction efficiency results showed CB-SPME to have lower percent recovery than LLE and SPE ( Table 1 ), yet demonstrated superior reproducibility and lower variability across replicates (%RSD), highlighting its consistency and accuracy ( Table 1 ). In both SPE and LLE extraction processes, the samples are actively forced through a column containing a sorbent (in SPE) or mixed with a solvent (in LLE), which selectively binds or dissolves desired compounds. This active process enhances interface interactions, often leading to higher recoveries of target compounds (Alvarez et al., 2004; Renaud et al., 2022; Tadić et al., 2022). However, this forced interactions with the sample matrix also introduces greater variability, as reflected in the higher % RSD values for SPE and LLE ( Table 1 ). Conversely, the passive nature of CB-SPME, where analyte adsorption depends on natural diffusion at the sampling interface, results in a slightly reduced recovery efficiency compared to SPE and LLE. This passive sampling approach avoids the need for active interactions with the matrix, leading to significantly lower % RSD values across all metabolites, highlighting the method’s reproducibility and reliability. Additionally, the inflated recoveries observed for some metabolites with SPE (e.g., 195% for genistein) and LLE (e.g., 159% for daidzein) suggest matrix effects or co-extraction of interfering compounds, which may compromise the accuracy of quantification (Moreno-González et al., 2017; Cortese et al., 2020; Williams et al., 2023). In contrast, CB-SPME’s consistent recovery rates, despite being lower, offer a more controlled and reproducible extraction process, particularly beneficial for studies focusing on relative metabolite profiling over time. An additional notable advantage of CB-SPME is its ability to use multiple blades simultaneously, in contrast to one-by-one sample processing. CB-SPME enables faster sample preparation and extraction ( Table 2 ) while reducing solvent use, making it a more sustainable alternative aligned with green chemistry principles (Vrana et al., 2005; Gómez-Ríos et al., 2018; Kasperkiewicz and Pawliszyn, 2021). Consistent with these performance metrics, both the PCA plot ( Figure 2A ) and the base-peak chromatograms ( Figure 2B ) show that CB-SPME and SPE share highly similar metabolite fingerprints, whereas LLE yields a distinct profile that may reflect the method’s broader affinity for hydrophobic or late-eluting compounds. While CB-SPME showed lower absolute recovery in standard solutions, its metabolite profile closely aligned with that of SPE in real root exudate samples ( Figures 2A, B ). This is likely due to both methods using similar sorbent chemistries, which select for overlapping compound classes. Thus, despite lower yield, CB-SPME reliably captures a representative and reproducible profile, supporting its use for in situ biological sampling.

In root exudate, (iso)flavonoids, play a crucial role in plant-microbe interactions (Dhaubhadel et al., 2003; Baerson and Rimando, 2007; Cesco et al., 2010; Tian et al., 2021). Our targeted metabolomics using discrete sampling to investigate the effect of the three methods on the yield of various isoflavonoids in root exudate showed CB-SPME method to have equivalent detection and sensitivity compared to SPE and LLE ( Figure 3 ). Notably, their increased abundance in A. euteiches-infected samples highlights their role as stress-responsive defense compounds, consistent with previous findings identifying isoflavonoids as reliable indicators of plant immune responses (Ayabe et al., 2010; Shin et al., 2014).

Discrete sampling introduces bias by capturing only a specific moment in the dynamic process of root exudation, potentially missing the temporal variability and full spectrum of metabolites present (Vranova et al., 2013; Escolà Casas and Matamoros, 2021). In contrast, several studies have implemented passive sampling, primarily in aquatic environments, and found it to be superior, identifying more compounds with better isotopic pattern matches, indicating higher contaminant concentrations in extracts (Alvarez et al., 2004; Vrana et al., 2005; Lissalde et al., 2011; Tadić et al., 2022). While the use of passive sampling in the study of root exudates is a relatively new approach, one study utilized SPME fiber for passive sampling of root volatiles from glasshouse-grown broccoli in situ (Deasy et al., 2016). However, the insertion of SPME fibers into the vicinity of growing roots may perturb root behavior and alter the soil’s physicochemical properties. This is further complicated by the fibers’ fragility and susceptibility to damage during collection. The use of hydroponic systems allows for direct access to exudates without soil interference, minimizing root disturbance and enhancing the accuracy of studying root exudate dynamics (Strehmel et al., 2014). This approach preserves the rhizosphere environment while enabling precise temporal analysis of metabolites.

Isoflavonoid pisatin, being a phytoalexin, is typically produced by pea plants as a part of their immediate defense mechanism against pathogens (Borejsza-Wysocki et al., 1997; George and Vanetten, 2001; Wu and Vanetten, 2004; Cannesan et al., 2011). The initial peak of pisatin observed at 2 days post-infection suggests an active and robust defense response ( Figure 4 ), while the subsequent decline in levels could indicate a potential successful suppression of the pathogen, leading to reduced production of the phytoalexin, or an adaptation of the pathogen to the plant’s defense mechanisms (Miao and Vanetten, 1992; George and Vanetten, 2001). Pisatin accumulation monitored in this study illustrates CB-SPME’s utility in real-time, in situ monitoring of defense metabolite fluctuations, capturing biologically relevant temporal data. Notably, while our experiment was conducted in a hydroponic setup, the CB-SPME platform, as previously shown is compatible with both liquid and solid-phase systems. Thus, allowing for expansion to soil and its future applicability for root exudate monitoring in more natural environments.

The passive nature of CB-SPME has proved to be very effective in our temporal targeted metabolomic analysis, opening doors for further root-specific research. This integrated approach minimizes sample handling, reduces the risk of metabolite loss or degradation, and enhances reproducibility in metabolomic profiling. Importantly, it enables detailed investigation of plant–pathogen interactions under conditions that preserve the natural rhizosphere and minimize experimental disturbances. These insights have the potential to lead to breakthroughs in understanding how plants responds to pathogens at the molecular level, and the development of crops that are more resistant to diseases.