Root uptake of umbelliferone enhances pea ’ s resistance against root-knot nematodes

Coumarins are secondary plant metabolites which play a key role in plant – plant and plant – microbe interactions. In particular, they are highly involved in environmental stress responses. Coumarins can transfer from plants producing coumarins to non-coumarin-producing neighbouring plants. As in vitro studies have shown that coumarins are nematicidal, we hypothesized that this transfer may also result in enhanced resistance against plant parasitic nematodes in coumarin-receiving plants. To test if the uptake of coumarins in a non-coumarin-producing plant protects the plant against nematode attack, we incubated non-coumarin-producing pea seedlings in growth media with the coumarin umbelliferone for three weeks, after which the plants were transplanted into soil and inoculated with root-knot nematodes ( Meloidogyne incognita ). We quantified the coumarin content in pea organs and nematode root invasion 2, 4, and 6 weeks after transplantation. Umbelliferone was taken up by the pea roots and translocated to the shoots. As a result of metabolization, umbelliferone and its derived metabolites coumarin, scopoletin, and scopolin were detected in the plants. The root uptake of umbelliferone reduced root-knot nematode invasion significantly up to 4 weeks after the root exposure. Our results suggest that the root uptake and the transfer of bioactive compounds between plants can expand the understanding of plant – plant interactions.


Introduction
Plants exude a wide range of primary and secondary metabolites from their roots (Kong et al., 2018;Walker et al., 2003).Plant roots can take up root exudates of neighbouring plants and then translocate them into aerial parts of the receiving plant (Shajib et al., 2012;Trapp, 2004;Trapp and Legind, 2011).Bioactive root exudates may be allelochemicals, inhibiting the growth of neighbouring plants (Bais et al., 2004;Gniazdowska and Bogatek, 2005;Weir et al., 2004).Until recently, research mainly focused on the negative effects of bioactive exudates in the receiving plant.Meanwhile, bioactive compounds may also act as defence compounds against microbial pathogens and herbivores (Hu et al., 2018) and modulate the root microbiome (Kudjordjie et al., 2019;Sikder et al., 2021).We have shown that the uptake of bioactive compounds synthesized by other plant species can strengthen the receiving plant's defence against root parasitic nematodes (Hama et al., 2024).Thus, plant metabolites are highly involved in plant-plant, plant-pest, and plant-microbe interactions (Chagas et al., 2018;Stringlis et al., 2019), and we are beginning to realize that the transfer of bioactive compounds between plant species may play a hitherto overlooked role in plant defence.
Root-knot nematodes (Meloidogyne spp.) are plant parasitic nematodes.Due to their ubiquitous distribution, wide host range, and disruption of plant growth, they are considered among the most important plant parasitic nematodes (Jones et al., 2013).Apart from the adult male, the second stage juvenile (J2) is the only mobile life stage.The J2 hatches from the egg and moves towards the host plant's root, penetrating the root at the tip from where it moves to the vascular tissues.The J2 establishes a permanent feeding site in the vascular cylinder, where it attains an immobile or sedentary lifestyle.Via two more juvenile stages the nematode develops into adulthood.The adult female remains sedentary depositing eggs in a gelatinous egg mass either inside the root or into the soil, whereas the adult male regains mobility and migrates from the root to mate.
Root-knot nematodes can cause substantial loss in many crop species, including beans and peas, and the damage or yield loss is more profound when the plants are under additional stress (e.g.drought) (Bridge and Starr, 2007;Sharma and Baheti, 1992).For instance, in pea, Meloidogyne species damage and reduce the root system, limiting root penetration to deeper soil layers (Grünwald et al., 2004;Sharma and Baheti, 1992;Sharma and Tiagi, 1990).Pea plants do not synthesize umbelliferone but are able to take up umbelliferone (Hijazin et al., 2019).Having bioactive properties, we hypothesize that root uptake of umbelliferone assists the receiver plant, e.g.pea, in suppressing microbial pathogens and herbivores, e.g., root-knot nematodes.To test this hypothesis, we supplemented pea (Pisum sativum L.) growth media with umbelliferone, quantified umbelliferone and other coumarin transformation products in pea tissues, and examined the effect of umbelliferone uptake on rootknot nematode (M.incognita) root invasion.This study thus provides new insight into the impact of root-absorbed natural metabolites on plant susceptibility to root-knot nematode infestation.

Experimental setup
We designed an experiment to elucidate the effect of pea root umbelliferone uptake on root-knot nematode invasion of pea roots.To avoid confounding effects of umbelliferone in the soil on the nematodes, root exposure to umbelliferone was separated in time and space from the exposure to nematodes (Fig. 1).We thus incubated pea seedlings for three weeks in MS medium spiked with umbelliferone to allow root uptake.After the incubation period, we transplanted the plants to soil and inoculated the soil with infective juveniles (stage J2) of the rootknot nematode M. incognita.
Pea (P.sativum L. cv.ESO, (DLF, Roskilde, Denmark)) seeds were sterilized before germination by rinsing the seeds with 70 % ethanol and 1.5 % sodium hypochlorite for 1 and 4 min, respectively, followed by washing with sterilized Milli-Q water.We pregerminated the sterilized seeds on MS medium (0.4 % Murashige and Skoog basal salt mixture, 0.3 % phytagel, and 2 % sucrose, adjusted pH to 5.8) in a petri dish, placed in a growth chamber (day and night temperatures of 20/16 • C, 12 h light:12 h dark) for one week.We transferred the germinated seedlings to Phytatray II boxes (Sigma-Aldrich Intl GmbH, Schnelldorf, Germany) (6 plants per box), containing another MS medium (0.22 % Murashige and Skoog basal salt mixture, 0.5 % sucrose, 0.3 % phytagel, adjusted pH to 5.8), which was either spiked with 100 μM umbelliferone (final concentration) or not spiked (control).For spiking, we added aliquots of umbelliferone to empty Phytatray II boxes and left to evaporate.Hot (40 • C) MS medium was then added to the Phytatray II boxes and stirred to dissolve the umbelliferone and homogenize the solution.Phytatray II boxes with the MS medium solution were left in a laminar flow cabinet to solidify.In the control treatment, the MS medium was not spiked.The Phytatray II boxes were placed in a growth chamber (day and night temperatures of 20/16 • C, 12 h light:12 h dark) for 3 weeks.
We transplanted the three weeks old pea plants (from the umbelliferone and control treatments) into pots filled with 0.7 kg of soil (fw).The soil was prepared by mixing sandy-clayey soil from an arable field in Flakkebjerg, Denmark, with washed sand particles (particle size 0.3-1 mm) (Dansand, Braedstrup, Denmark) (2:3, w/w).Characterization of the field soil is summarized in Supplementary Table S1.The pots were transferred to a greenhouse and arranged using a completely randomized design per treatment, under a 12 h:12 h light:dark photoperiod at 22-24 • C temperature.We watered the pots as needed with a 1 % fertilizer solution (PIONER BASIS NPK 14-2-23 + Mg + MIKRO (Brøste, Copenhagen, Denmark) and YaraLiva® CALCINIT™15.5-0-0(Yara Florida, USA)).
For LC-MS/MS analysis, we harvested pea plants 0, 2, 4, and 6 weeks after the umbelliferone root exposure, i.e. number of weeks after transfer from the Phytatray boxes to soil, and also collected MS media and soil samples.The roots were washed with distilled water and separated from the shoots.Upon sampling, the root, shoot, MS medium, and soil samples were immediately immersed in liquid nitrogen to prevent any enzymatic reaction before being stored at − 80 • C until processing.Three different setups were investigated: pea plants exposed to umbelliferone, control plants (not supplemented with umbelliferone), and soil control without plants.For the umbelliferone treatment, we sampled three replicate plants, and for the control treatment, we sampled six replicate plants.We also sampled three replicates of the umbelliferone-spiked MS media and soil samples (from all treatments: umbelliferone, control, and soil control without plant).In total, we collected 105 samples, comprising 36 root, 36 shoot, 27 soil, and 6 MS medium samples (summarized in Supplementary Table S2).
To investigate root-knot nematode invasion of the roots, we prepared additional pots with pea plants (transferred from umbelliferone spiked and control treatments), which we inoculated with M. incognita.The inocula of M. incognita infective second-stage juveniles (J2s) were prepared from a culture maintained on tomato plants (cv.Moneymaker).We blended the tomato roots in 1.5 % NaOCl and collected the eggs on a sieve (25 μm).We transferred the eggs to a Baermann tray and collected freshly hatched J2s daily for a week.One set of plants were inoculated with J2s 1, 3 and 5 weeks after the plants were transferred to soil from the control or umbelliferone spiked media, respectively.We pipetted 1500 J2s suspended in 5 mL tap water to the soil around the stems at each inoculation.We harvested the plants one week after inoculation, i. e. 2, 4, and 6 weeks after umbelliferone root exposure, respectively.Upon harvest, the roots were washed with distilled water and then carefully separated from the shoots.To visualize the nematodes that had penetrated the pea roots, we stained the roots with an acid-fuchsin solution and counted the number of stained J2s per root system under a stereomicroscope (Hooper et al., 2005).Three different setups were investigated: pea plants exposed to umbelliferone, control plants, and control plants without nematode inoculation.At each sampling time, we quantified nematode invasion in five replicate plants of each treatment, except at week 6 after exposure to umbelliferone, where the noninoculated control was omitted, amounting to 40 samples in total (summarized in Supplementary Table S3).

Extraction of plants and soil
For LC-MS/MS analysis, we extracted freeze-dried homogenized plant material (20 mg) with 1 mL of 80 % methanol containing 1 % acetic acid in an Eppendorf tube by sonication for 45 min.The tube was vortexed for 1 min, and centrifuged (Sigma 1-14 K micro-centrifuge, SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany) for 10 min at 4500g.The supernatant was collected into a 4 mL amber glass tube.The plant material was extracted for the second time by 1 mL of methanol+1 % acetic acid following the procedure described above.Subsequently, we combined the supernatants, diluted with Milli-Q H 2 O (1:1), and filtered through a 0.22 μm PTFE syringe filter into an HPLC glass vial.The vials were stored at − 20 • C prior to analysis by LC-MS/ MS.
Soil samples were extracted by the method described by Pedersen et al. ( 2011) with minor modifications.Freeze-dried soil was homogenized to a powder in a Tube Mill 100 control blender (Staufen, Germany).The soil was extracted using a Dionex ASE 350 Accelerated Solvent Extractor (ASE) (Hvidovre, Denmark).Extraction ASE cells with soil were prepared as follows: a cellulose filter was placed at the bottom of the cell, followed by the addition of 5 g of inert Ottawa sand, 5 g of the soil sample, another 5 g of inert Ottawa sand, and finally another cellulose filter.The remainder of the cells were filled with glass beads.The samples were extracted using the following settings: temperature, 80 • C; heat, 5 min; static time, 3 min; cycles, 4; rinse volume, 60 %; purge, 60 s.35 mL extraction solvent (80 % methanol and 1 % acetic acid (v/v)) was used.The procedural blank for the method was prepared the same way as the samples, but another 5 g of inert Ottawa sand was used instead of soil.

LC-MS/MS analysis
We analysed extracted samples on an Agilent 1100 HPLC system coupled with a 3200 QTRAP mass spectrometer (AB SCIEX, Foster City, California, USA) in positive ionization.The separation was obtained in one chromatographic run using a 250 mm × 2 mm id 4 μm Synergi Polar RP-80 Å column (Phenomenex, Macclesfield, United Kingdom).25 μL of each sample were injected into the pre-heated column at 30 • C, with a flow rate of 200 μL/min.The mobile phase consisted of eluent A (100 % MilliQ +20 mM acetic acid) and eluent B (100 % methanol +20 mM acetic acid), used in a gradient elution mode: 0-2 min 90 % A, 2-4 min decreasing to 70 % A, 4-10 min decreasing to 40 % A, 10-20 min decreasing to 1 % A, 20-23 min staying at 1 % A, 23-23.5 min increasing to 90 % A, and 23-30 min equilibrating at 90 % A. In the QTRAP mass spectrometer, two multiple reaction monitoring (MRM) transitions were used to monitor coumarins (Table 1).Quantification ions were used to determine the concentrations, while qualification ions served as a quality assurance for confirming the identity of the compounds.For quantification, we prepared calibration curves based on coumarin standard solutions (listed in Table 1) in the range of 0.39-800 μg L − 1 by plotting measured analyte peak areas against corresponding analyte concentrations, using quadratic regression and 1/x weighing for each curve.Samples were diluted when their concentrations fell outside the range of the calibration curve.To check the instrumental response and drifts in the MS/MS, a standard solution (50 μg L − 1 ) was analysed with every 15 injections.In addition, at least one instrumental blank sample was analysed with every 15 injections; no coumarins were found in instrumental blanks.During the data processing, the signal of lowest standard solution was used as baseline, to remove the background noise.Peaks were included when the quantifier/qualifier ion relation for sample peaks was in a range of ±20 % of the quantifier/qualifier ion relation of standards and if they were detected in three or more of the six replicates.Analyst Software (version 1.7.1,AB Sciex Foster City, California, USA) was used for instrument control, data acquisition, and subsequent quantifications.

Statistical analysis
To determine whether nematode invasion (absolute number of nematodes per root system) and plant dry biomass were significantly different between control and umbelliferone-treated plants, we performed two-way ANOVA using the treatments and sampling time as categorical factors.We tested for homogeneity of variance using Levene's test.Where necessary, data were log-transformed to obtain homogeneity of variance.Statistical analyses were performed using OriginPro software 9.6 (OriginLab Inc., Massachusetts, USA).

Root uptake experiment
We grew pea seedlings for 3 weeks in MS medium spiked with umbelliferone to allow the seedlings to take up umbelliferone through the roots, after which we transplanted the seedlings to soil-filled pots (Fig. 1).Coumarin content in pea tissues and root-knot nematode invasion were quantified in the transplanted plants up to 6 weeks after transplantation.By transplanting the plants from MS medium (the umbelliferone source) to the umbelliferone-free soil, we avoided any direct effects of umbelliferone in the soil or the medium on the nematodes.

Coumarins in pea plant
We detected and quantified umbelliferone and other coumarins in the pea roots (Fig. 2) and shoots (Fig. 3) at 0, 2, 4, and 6 weeks after transplantation from the umbelliferone-spiked MS medium to soil showing that umbelliferone was absorbed by the pea roots and translocated to the shoots.At week 0 after umbelliferone root exposure, the root concentration of umbelliferone was 43 μg g − 1 dry weight (dw), and it declined to 2.6 μg g − 1 dw in week 6.Following the uptake of umbelliferone in the roots, we detected scopoletin and scopolin (Fig. 2).Umbelliferone was methoxylated to form scopoletin, which subsequently was glycosylated to form scopolin (Hijazin et al., 2019).
In the pea shoots (Fig. 3), the umbelliferone concentration was 1.2 μg g − 1 dw at week 0 after root umbelliferone exposure, and the concentration decreased to 0.06 μg g − 1 dw at week 4 after root umbelliferone exposure.Umbelliferone was not detected at week 6.The transformation of umbelliferone in the shoots was a reduction to coumarin (Fig. 3).The profile of the coumarins differed between pea tissues; in the roots, we found umbelliferone, scopoletin and scopolin, while we only found umbelliferone and coumarin in the shoots.Importantly, we did not detect umbelliferone or any other coumarin compound in roots or shoots of the control plants incubated in an umbelliferone-free MS medium.Likewise, all soil samples were free from umbelliferone and other coumarins.
The maximum concentration of umbelliferone in the roots detected at week 0 after root exposure was approximately 200 % of the initial concentration in the MS media.As the concentration in the plant was higher than in the medium, an active transport mechanism must have been involved in the root uptake of umbelliferone.The amount of root uptake depends on the chemical properties of umbelliferone, particularly the log P-value.Other studies documented that low log P-values facilitate root uptake of a compound (Limmer and Burken, 2014;Schriever and Lamshoeft, 2020), and that chemical compounds with log P values lower than 3 were taken up by roots through diffusion (Limmer and Burken, 2014;Trapp, 2004).The log P-value of umbelliferone is 1.6, which is in the range of passive root absorption.However, we have found that major passive uptake was seen in plants treated with compounds with log P-values <0.3 (Hama et al., 2022).
Pea plants thus absorbed umbelliferone from the growth medium.This thus suggests that umbelliferone exuded from umbelliferonesynthesizing plants to soil in a co-cropping system could be taken up by the roots of neighbouring heterospecific plants.However, in pea roots and shoots, umbelliferone concentrations decreased substantially with time; at weeks 2 to 6 after root exposure either no (in shoots) or trace concentrations (in roots) were detected.The continuous decline of umbelliferone concentrations may be due to metabolization.In addition, the increasing plant biomass might contribute to the dilution of umbelliferone concentrations in the plant.To which extend umbelliferone will transfer between heterospecific plants in co-cropping systems will depend on the bioavailability of umbelliferone in soil.The impact of soil physicochemical parameters on the bioavailability of umbelliferone is not well elucidated, but umbelliferone sorption was higher in acid soils than in alkaline soils (Real et al., 2019).Further investigations are thus needed to quantify umbelliferone transfer between heterospecific plants in a range of soil types and to clarify how the transfer is influenced by soil physicochemical parameters.

Impact of umbelliferone on nematode invasion and plant growth
To understand the impact of umbelliferone uptake in the pea roots on root-knot nematode invasion, we inoculated pea roots with infective juveniles (stage J2) of the Southern root-knot nematode (M.incognita) one week before each of the sampling days.The root exposure to umbelliferone impacted M. incognita invasion with time after exposure (two-way ANOVA treatment p = 0.02, time p < 0.0001, treatment x time p = 0.0001).Two weeks after exposure, the presence of umbelliferone reduced M. incognita invasion significantly (Fig. 4).In control roots, nematode invasion was five-fold higher (mean 147 J2s per root) than the umbelliferone treated roots (mean 30 J2s per root).The impact of root exposure to umbelliferone was still significant 4 weeks after root exposure, where the mean number of J2s were two times higher in control than in umbelliferone exposed roots.Four weeks after root exposure, the plants were 7 weeks old and had reached the flowering stage.Generally, the susceptibility to root-knot nematodes is highest at seedling and early vegetative stages (Griffin and Hunt, 1972;Kayani et al., 2018).With reduced nematode invasion up to 4 weeks after exposure to umbelliferone, the pea plants thus gained enhanced resistance during the most susceptible growth stages.
However, there was no effect at 6 weeks after umbelliferone root exposure, where coumarin concentrations had also decreased considerably (Fig. 2).Thus, this suggests that the reduction of nematodes in roots depended on the umbelliferone root concentration, where higher concentrations suppressed nematode invasion at 2-4 weeks after umbelliferone exposure.In addition to the suppressive effects of umbelliferone on nematodes, the reduction of nematode invasion may also reflect umbelliferone-induced immune responses in the pea plants.For example, reactive oxygen species (ROS) play a central role in plant immune responses, and umbelliferone is reported to induce the accumulation ROS in root tips of lettuce (Lactuca sativa L.) plants (Pan et al., 2015).This may explain that umbelliferone-treated plants are better protected against nematode invasion at 2 and 4 weeks after umbelliferone exposure, where umbelliferone/coumarins were considerably lower than at week 0. The roots were free from nematodes in pots that were not inoculated with M. incognita.
The enhanced resistance against M. incognita invasion in plants that had taken up umbelliferone corroborates other studies in vitro, which demonstrated that umbelliferone and other coumarins were antagonistic against root-knot nematodes (Caboni et al., 2015;Wuyts et al., 2006).Further, root extracts of Stellera chamaejasme Linn.with high umbelliferone content were highly toxic against pine wood nematodes (Bursaphelenchys xylophilus), with a lethal concentration (LC 50 ) of 3.3 μM after 72 h exposure (Cui et al., 2014;Feng et al., 2022).In addition, umbelliferone was repellent against the plant parasitic nematode Radopholus similis in vitro at concentrations down to 257 μg mL − 1 (Wuyts et al., 2006).In the studies discussed above, the biological activities of umbelliferone/coumarins were tested in vitro.This study is thus the first to report that the root uptake of umbelliferone suppressed plant parasitic nematodes (M.incognita) invasion in pea roots grown in soil, supporting the nematicidal activity of umbelliferone in a plant-soil system.
Future studies will elucidate if co-cultivation of e.g.pea and umbelliferone synthesizing plants will similarly result in transfer of umbelliferone and enhanced protection against root-knot nematodes.If that is the case, we anticipate a continuous transfer of umbelliferone to the receiver plant and thus a continuously or prolonged enhanced protection against root-knot nematodes during the growing season.In addition, it will be very interesting to clarify if root uptake of umbelliferone also reduces M. incognita development and reproduction, thus adding value to the enhanced invasion resistance of the receiver plants.Further studies are also needed to assess to which extent umbelliferone/ coumarin uptake can supplement pea plant defence against a wider array of pests and pathogens.It is also worth considering if umbelliferone/coumarins have un-desirable effects on non-target organisms, including non-herbivorous soil and rhizosphere nematode species, many of which often exert positive effects on plant growth.For instance, microbial feeding nematodes generally stimulate mineralization of plantavailable nutrients (Rønn et al., 2012), and entomopathogenic nematodes may control populations of root herbivorous insects (Sikder and Vestergård, 2020).
In a similar experiment we showed that root uptake of benzoxazinoids in clover roots reduced M. incognita J2 invasion (Hama et al., 2024).It thus appears that root uptake of plant metabolites produced by other plant species could be a general mechanism of enhancing defence capacity in the receiving plant.This could be exploited in crop production, where co-cropping of plant species producing complementary and transferrable defence metabolites potentially could improve the inherent pest resistance of cropping systems.
Overall, the root and shoot biomass increased with time (Fig. 5).Despite the variation between the treatments, the umbelliferone exposure did not affect biomass significantly (Two-way ANOVA treatment p = 0.26 for root biomass and p = 0.49 for shoot biomass).Umbelliferone is an allelopathic metabolite, which can inhibit the growth of neighbouring plants.Other studies reported that for example root structure and plant biomass of lettuce (Lactuca sativa L.) plants and several weed species were effectively inhibited when exposed to umbelliferone (Kato-Noguchi et al., 2023;Kupidlowska et al., 1994;Pan et al., 2015;Yan et al., 2016).Further, umbelliferone is one of the causal factors for the allelopathic activity of S. chamaejasme, which explains its rapid and invasive spread (Guo et al., 2015;Niro et al., 2016).However, the effect of umbelliferone on plant biomass is species dependent.In our case, umbelliferone did not suppress pea plant growth.Similarly, Kupidlowska et al. (1994) reported that other coumarins (coumarin and xanthotoxin) did not reduce garden pea growth (Kupidlowska et al., 1994).
Thus, we suggest that between-plant species transfer of bioactive compounds may be an important -but hitherto neglected -mechanism in plant-to-plant interactions with significant importance for plant defence

Fig. 1 .
Fig. 1.Timeline of the experimental setup of pea root exposure to umbelliferone and sampling throughout the experiment.

Fig. 2 .
Fig. 2. Mean concentration (μg g − 1 dw) of coumarins in pea roots after root exposure to 100 μM of umbelliferone.Pea plants were grown for 3 weeks in MS medium spiked with umbelliferone, and then transplanted to soil for 6 weeks.Column bars represent mean ± standard deviations, n = 3.

Fig. 3 .
Fig. 3. Mean concentration (μg g − 1 dw) of coumarins in pea shoots after root exposure to 100 μM of umbelliferone.Pea plants were grown for 3 weeks in MS medium spiked with umbelliferone, and then transplanted to soil for 6 weeks.Column bars represent mean ± standard deviations, n = 3.

Fig. 4 .
Fig. 4. Invasion of M. incognita J2 in pea roots 2, 4 and 6 weeks after root exposure to 100 μM umbelliferone.The effect of umbelliferone exposure on M. incognita J2 invasion depended on the time since exposure (Two-way ANOVA treatment p = 0.02, time p < 0.0001, treatment x time p = 0.0001).

Fig. 5 .
Fig. 5. Dry biomass of pea plant roots and shoots.Plants were grown is MS media spiked with 100 μM umbelliferone or in unspiked media (Control) for 3 weeks, and then transplanted to soil.Values plotted mean ± standard deviation (SD) (n = 3 for umbelliferone, and n = 6 for control treatment).There were no effects of umbelliferone exposure at any of the timepoints (Two-way ANOVA treatment p = 0.25 for root biomass and p = 0.49 for shoot biomass).

Table 1
Multiple reaction monitoring (MRM) transitions and compound-dependent parameters used for coumarin quantifications during LC-MS/MS analysis.