Dual and single-species nematode infections distinctly modulate defense metabolism in Brassica nigra roots

Dual and single-species nematode infections distinctly modulate defense metabolism in Brassica nigra roots | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dual and single-species nematode infections distinctly modulate defense metabolism in Brassica nigra roots Jessil Ann Pajar, April Lyn Leonar, Pius Otto, Franziska Sabine Hanschen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6835102/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Sep, 2025 Read the published version in Journal of Chemical Ecology → Version 1 posted 9 You are reading this latest preprint version Abstract Plant roots are exposed to various organisms that significantly impact plant productivity. Plant-parasitic nematodes (PPNs) such as Meloidogyne spp. and Pratylenchus spp. are microscopic roundworms that damage several crops. In natural populations, Meloidogyne spp. and P. penetrans were found to infest black mustard ( Brassica nigra ) plants simultaneously. Considering their different feeding strategies and contrasting effects on plant defense responses, we hypothesized that dual infection may affect each nematode’s performance via changes in the root metabolome. Using untargeted and targeted metabolomics, we evaluated how single and dual nematode infections affected B. nigra root metabolome. We combined these metabolic data with measures of early infection success. At three days post-inoculation, dual infection increased M. incognita penetration success, while that of P. penetrans remained unaffected. Compared to single-species infections, dual infections resulted in distinct root metabolic changes by reducing indole glucosinolates (GSL), gluconasturtiin, lignans, and phenylpropanoid levels. Dual and single-species infections affected different GSL classes. Sinigrin and its breakdown products increased in response to P. penetrans, while M. incognita infection increased gluconasturtiin and 2-phenylethyl ITC . This shows that plant defense response to dual nematode infection differ from those of single species, which has consequences to the early infection success of each nematode species. glucosinolates nematode-nematode interactions plant defense root metabolome simultaneous herbivory systemic induced responses Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Plant-parasitic nematodes (PPNs) are root-feeding roundworms that pose significant threats to global agriculture. Several PPN species are causing extensive damage to crop plants (Nicol et al., 2011 ; Jones et al., 2013 ; Tileubayeva et al., 2021 ), with yield losses estimated at 173 billion USD worldwide (Kantor et al., 2022 ). Meloidogyne incognita and Pratylenchus penetrans are two prominent PPN species known for their detrimental effects on various crops (Jones et al., 2013 ; Kantor et al., 2022 ; Zhang et al., 2023 ). The root-knot nematode (RKN) M. incognita is a sedentary endoparasitic nematode. The infective juvenile (J2) invades the roots by moving in-between cells until it reaches the vascular cylinder and induces the formation of giant cells. Thereafter, it becomes sedentary throughout its feeding and reproductive stages (Kyndt et al., 2012a ; Bartlem et al., 2014 ; Rutter et al., 2022 ). The giant cells serve as nutrient sinks, diverting resources away from other plant tissues and impairing its growth and productivity (Kyndt et al., 2013 ; Rutter et al., 2022 ; Mitchum et al., 2023 ). Additionally, M. incognita secretes effectors to modulate its host's immune responses and facilitate parasitism (Gheysen and Mitchum, 2011 ; Eloh et al., 2016 ; Rutter et al., 2022 ). Pratylenchus penetrans , on the other hand, is a migratory endoparasitic nematode that feeds by puncturing individual cells and withdrawing nutrients as they move through the roots (Sijmons et al., 1994 ; Fosu-Nyarko and Jones, 2016 ). The effectors secreted by P. penetrans enable mobility, causing tissue destruction due to, for example, cell wall degrading enzymes (Vieira et al., 2018 ). Unlike RKNs, P. penetrans does not induce the formation of giant cells, but it can cause extensive root damage leading to root necrosis, reduced nutrient and water uptake, as well as render the infected roots vulnerable to secondary microbial infections (Sijmons et al., 1994 ; Fosu-Nyarko and Jones, 2016 ). Several studies showed that plants can recognize and respond to nematode infections through various defense mechanisms. This includes activating defense-related genes and synthesizing secondary metabolites (Hofmann et al., 2010 ; Kyndt et al., 2012a ; Desmedt et al., 2020 ). Studies on related PPNs demonstrated that migratory and sedentary endoparasitic nematodes regulate plant defenses differently (Lohmann et al., 2009 ; Kyndt et al., 2012b ). In rice, early root infection by the migratory endoparasitic nematode Hirsmaniella oryzae up-regulated biotic stress-responsive genes, inducing oxidative stress and programmed cell death. In contrast, early infection by the sedentary endoparasite, Meloidogyne graminicola , suppressed root defense-responsive genes (Kyndt et al., 2012a ). Brassica spp. plants are frequently challenged by PPNs (Liébanas and Castillo, 2004 ; Mukhopadhyay and Roy, 2006 ; Hol et al., 2016 ). In a survey by Hol et al. ( 2016 ), Meloidogyne spp. and Pratylenchus spp. were found to infest field-grown black mustard plants, Brassica nigra . Brassica plants are known to deploy glucosinolates as defense response against nematodes (van Dam et al., 2018 ; Desmedt et al., 2020 ). Glucosinolates (GSLs) are the main defense compounds of plants in the order Brassicales (Tsunoda et al., 2017 ; Touw et al., 2020 ). They are β- D -thioglucosides that can be classified by differences in their side chains and are grouped into aliphatic, benzenic, or indolic GSLs (Kliebenstein et al., 2001 ; Agerbirk and Olsen, 2012 ). Analyzing GSL profiles of nine GSL-producing plants including B. nigra , Tsunoda et al. ( 2017 ) showed that root GSL concentrations are significantly higher than in the shoots. Other than shoots, roots contain high levels of gluconasturtiin (2-phenylethyl GSL) (Tsunoda et al., 2017 ; Touw et al., 2020 ; Sontowski et al., 2022 ), which serves as defense against nematodes (Potter et al., 1998 ) and insect herbivores, such as Delia radicum and D. floralis (Sontowski et al., 2022 ). Several GSLs, either applied as plant extracts or incorporated as biofumigants, can reduce PPN populations and associated symptoms (Ren et al., 2018 ; Yu et al., 2019 ; Dahlin and Hallmann, 2020 ; Eugui et al., 2022 ). Beyond the localized defense response, GSLs and other plant secondary metabolites are induced systemically in response to nematode infections, which correlated with altered performance of other herbivores feeding on the same plant (Hol et al., 2016 ; van Dam et al., 2018 ; Pajar et al., 2024 ; Touw et al., 2025). Plant-mediated interactions between nematodes and aboveground insects, such as aphids and caterpillars (Hol et al., 2016 ; van Dam et al., 2018 ; Mbaluto et al., 2020 ), and on root-feeding insects such as D. radicum , have been reported (Touw et al., 2025). However, most studies have focused on responses to infections by single PPN species. This overlooks the fact that in natural environments, multiple PPN species interact within the same host plant (Hol et al., 2016 ; Mateille et al., 2020 ). Sedentary and migratory PPN species may antagonize each other, whereby the population increase of one species suppresses the other (Gay and Bird, 1973 ; Chapman and Turner, 1975 ; Fontana et al., 2015 ). It has been suggested that such effects are caused by competition for feeding sites or resources (Mateille et al., 2020 ). Alternatively, antagonistic relationships between PPNs may be governed by plant-mediated mechanisms. For example, the phytohormones salicylic acid (SA) and jasmonic acid (JA) can be activated during PPN infections, which mainly depends on the PPN species interacting with the plant (Kyndt et al., 2012a ; van Dam et al., 2018 ; Gheysen and Mitchum, 2019 ). In general, SA is associated with systemic acquired resistance and is effective against biotrophic pathogens and sedentary PPNs (Bonnet et al., 2017 ; Gheysen and Mitchum, 2019 ). On the other hand, JA is involved in induced responses that are commonly more effective against necrotrophic pathogens and migratory PPNs (Bonnet et al., 2017 ). Commonly, JA and SA act antagonistically via phytohormonal crosstalk (Pieterse et al., 2009 ; Bonnet et al., 2017 ; Gheysen and Mitchum, 2019 ). When a plant is simultaneously infected by several PPNs, JA-SA crosstalk may lead to a modified metabolic response, as it was found for chewing and phloem-feeding insects aboveground (Bonnet et al., 2017 ). This also prompts the question of whether the plant's defense response against concurrent (dual) nematode infections differs from those activated during single-species infections. In this study, we examined the changes in the root metabolome of B. nigra when the plant is challenged by PPNs with contrasting feeding strategies: the migratory endoparasitic nematode P. penetrans , and the sedentary endoparasitic nematode, M. incognita. We hypothesized that the root metabolic changes in response to each nematode species are distinct due to their different feeding strategies. Moreover, we also analyzed root metabolic changes in response to the concurrent infection of M. incognita and P. penetrans. We hypothesized that simultaneous infection would attenuate the defense response in comparison to single-species infections. Thus, the performance of one or both nematodes would improve on plants with simultaneous or prior infection by the other nematode. To test these hypotheses, we performed untargeted metabolic analysis on root samples infected with each nematode species as well as on root samples with concurrent infection. Our results indicate that root metabolic changes in response to concurrent infection by M. incognita and P. penetrans are distinct from those observed in single-species infections, in particular for GSLs, lignans, and phenylpropanoids. Targeted analyses of GSLs and their breakdown products showed that sinigrin and allyl isothiocyanate (ITC) levels increased in P. penetrans -treated plants, while gluconasturtiin and 2-phenylethyl ITC marginally increased in M. incognita -infected plants. These GSLs and ITCs were reduced in MP-treated plants, along with indole GSLs, lignans and phenylpropanoids. Concurrent infection increased the number of M. incognita inside the roots, while P. penetrans numbers remained unaffected. However, fewer P. penetrans were found in the roots when inoculated two days after M. incognita . Our results highlight the importance of studying plant response to multiple nematode infections, as this may lead to changes in plant defense mechanisms that differ from those observed in single-species infections. MATERIALS AND METHODS Plant Growing Conditions Brassica nigra seeds were bulk-collected from a wild population at Elderveld, Arnhem, the Netherlands, in 2005. Prior to germination, the seeds were washed with 1% sodium hypochlorite solution and rinsed with ultrapure water. These clean seeds were germinated in water-soaked glass beads in plastic containers. The containers were covered with transparent plastic lids and kept in a climate chamber in a 16:8 (light: dark) photoperiod at 20:16° C (day: night). The seeds were germinated for 10 days before transplanting in sand pots (for the root material sampling) or Pluronic gel plates (for nematode-nematode interaction assay). The plant pots were prepared and maintained following the protocols described by van Dam et al. (2003). Before transplanting, each pot was filled with 2.5 l of dry, heat-treated sand (90 °C for 1 hour) and supplied with 200 ml tap water. The plants were grown in a greenhouse at 16:8-hour photoperiod, minimum light intensity 300 µmol m -2 s -1 ; average temperature 25 °C; 60-80% relative humidity. The plants were supplied with 100 ml half-strength 3P Hoagland solution weekly. The developmental stages of B. nigra were monitored following the universal BBCH scale (Lancashire et al., 1991). Nematode Cultures Pratylenchus penetrans was provided by the Plant Science Research Unit, Research Institute for Agriculture, Fisheries and Food (ILVO), Merelbeke, Belgium. The culture was maintained in carrot discs at 25 ± 1 °C. Meloidogyne incognita was provided by Bejo, Warmenhuizen, the Netherlands and was maintained on Solanum lycopersicum cv. ‘Moneymaker’ under greenhouse conditions (16:8 photoperiod at 25 ± 3° C). All infective stages of P. penetrans (juveniles and adults) and J2s of M. incognita were extracted from the cultures via modified Baermann technique (Hooper et al., 2005). A solution containing a specified number of nematodes in water-Tween20® solution (0.04% v/v) was prepared and used for inoculation. Greenhouse Ex P eriment Nematode inoculation To investigate nematode-induced changes in the roots, plants were assigned to the following treatment groups: control, M. incognita only (Mi), P. penetrans only (Pp), M. incognita + P. penetrans inoculated simultaneously (MP). Four-week-old (BBCH 32) B. nigra plants were inoculated with 2 ml of nematode suspension, each containing 200 nematodes in the infective stage in water-Tween20® solution. The same number of nematodes was applied in MP-treatments with 100 J2s of M. incognita and 100 infective stages of P. penetrans in 2 ml solution. Control plants were mock-inoculated with water-Tween20® solution. The nematode suspension was introduced into a small hole near the roots. Thereafter, 50 ml of tap water was added to facilitate nematode dispersal. The nematodes were allowed to infect for 10 days. A separate group of nematode-infected plants were left to grow for sixteen days more (total=26 days) to see the root galls and/or lesions, thereby confirming the success of nematode infection (Fig. S1). Root Sampling On the tenth day-post nematode inoculation (10 dpi), whole root samples were collected for metabolic (n = 3-5) and gene expression (n=3-4) analyses. Plants were carefully lifted from their pots, and the roots were washed under running water. Excess water was drained, and the samples were collected, flash-frozen in liquid nitrogen, finely pulverized and stored at -80 °C freezer until further use. Untargeted Metabolomics via LC-MS coupled with MS/MS Root metabolites were extracted using Liquid Chromatography-Time of Flight-Mass Spectrometry (LC-ToF-MS) according to Weinhold et al. (2022). Twenty milligrams of ground freeze-dried root samples were mixed with 1 ml extraction solution (25% acetate buffer, pH 4.8 and 75% HPLC-grade MeOH). The mixture was placed in ultrasonic bath (5 minutes, 30Hz) then centrifuged at 15,000 x g for 15 minutes at room temperature. The supernatant was transferred to a new Eppendorf tube while the pellet was re-extracted with 1 ml of extraction solution, placed in ultrasonic bath (5 minutes, 30Hz) and centrifuged again at 15,000 x g for 15 minutes at room temperature. The supernatant was combined with that from the first extraction, then centrifuged at 15,000 x g for 10 minutes. Afterwards, 200 µl of supernatant was transferred in an HPLC vial and was added with 800 µl of extraction solution. The LC-MS analysis was conducted on an UltiMate™ 3000 Standard Ultra-High-Performance Liquid Chromatography system (UHPLC, Thermo Scientific) with an Acclaim® Rapid Separation Liquid Chromatography (RSLC) 120 column (150 mm × 2.1 mm, particle size 2.2 μm, ThermoFischer Scientific). The detailed instrument settings and post-processing parameters are provided in Method S1. After processing and blank feature-subtraction, the dataset contained 5,919 features. The resulting features with MS/MS data were annotated using an in-house spectral library. All detected features were classified and formatted using SIRIUS/CANOPUS (Dührkop et al., 2019; Dührkop et al., 2021; Hyun Woo Kim et al., 2021) and MetIgel v.1.0 ©Smith and Schedl, 2021. Targeted Glucosinolate Analysis The glucosinolates (GSLs) were quantified via High-Performance Liquid Chromatography (HPLC, UltiMate™ 3000, Thermo Scientific) using 50 mg freeze-dried ground root samples following the protocol by Grosser and van Dam (2017). The acquired data was further processed in Chromeleon 7.2 SR5 MUa (9624; Thermo Fisher Scientific, Waltham, MA, USA). Quantification of GSL Breakdown Products Glucosinolate breakdown products were extracted following Hanschen and Schreiner (2017). Briefly, 25 mg of freeze-dried ground root sample was weighed into extraction vials and left to hydrolyze with 250 µl ultrapure water for 1 hour. Dichloromethane (DCM, 2 ml) were added along with 100 µl DCM containing 0.2 µmol benzonitrile (internal standard). The solution was shaken and centrifuged. The resulting DCM extract was dried over Na 2 SO 4 . The extraction was repeated twice, adding only 1.5 ml DCM to the last two sets. The extracts were combined and reduced under nitrogen steam to 300 µl. The GSL breakdown products were quantified via Gas Chromatography-Mass Spectrometry (GC-MS, Agilent 7890 A Series GC System, Agilent Technologies) equipped with an Agilent 7683 Series Autosampler, an Agilent 7683B Series Injector and an Agilent 5975C inert XL MSD, using the settings as in (Hanschen, 2024). Gene Expression Analysis Representative GSL biosynthesis and transport genes, and genes involved in phytohormone-mediated defenses were analyzed to assess the involvement of the respective processes in plant-nematode interactions. The genes and their short descriptions are as follows: CYP83A1 (CYTOCHROME P450, FAMILY 83, SUBFAMILY A, POLYPEPTIDE 1) is involved in the biosynthesis of aliphatic and benzenic GSLs. CYP79B2 (CYTOCHROME P450, FAMILY 79, SUBFAMILY B, POLYPEPTIDE 2) is involved in the conversion of tryptophan to indole-3-acetaldoxime, a precursor to IAA and indole GSLs; MYB122 (transcription factor MYB122) is known to regulate indole GSL biosynthesis; PR1 (pathogenesis-related protein 1) is a salicylic acid-responsive gene; PAL1 (phenylalanine ammonia-lyase 1) involved in the first reaction in the biosynthesis of secondary metabolites from L-phenylalanine; ERF1 (ethylene response factor 1) encodes a transcription factor that can be activated by ethylene or jasmonates. The list of primer sequences used in this experiment is given in Table S1. Total RNA was extracted from 100 ± 5 mg ground frozen root tissue following a protocol adapted from Oñate-Sánchez and Vicente-Carbajosa (2008) as described in detail by Touw et al. (2020). The quality of DNAse I (Thermo Scientific, Waltham, MA, USA)-treated RNA was visually evaluated by gel-electrophoresis and by measurement of 260/230 nm and 260/280 nm absorbance ratios using a NanoPhotometer® P330 (Implen, Munich, Germany). Stable cDNA was synthesized from 4 μg purified total RNA using Revert Aid H minus reverse transcriptase (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The samples were incubated at 42 °C for 60 min, 50 °C for 15 min, and finally, 70 °C for 15 min in a thermal cycler (Techne, Stone, UK). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using the CFX384 Real-time system (BioRad, Munich, Germany) with gene-specific primers (Table S1). The qPCR conditions were: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. Three technical replicates were analyzed per gene for each biological replicate (n= 3-4). The data was normalized to the average expression of the housekeeping genes GAPDH and TIP41 . The relative expression of target genes was calculated using the 2 -ΔΔCT method as described in Livak and Schmittgen (2001). Nematode-nematode Interaction Assay Root attraction assays were performed using a Pluronic gel medium, which simulates the three-dimensional soil environment (Wang et al., 2010). Following the protocol by Wang et al. (2009), the nematodes were mixed evenly into the Pluronic gel (Pluronic F-127, Sigma-Aldrich). One ml of gel was poured onto 3-cm Petri plates with 100 nematodes per plate. The plates were incubated at 27 °C for the gel to slightly solidify. The treatments were as follows: control (+ water), M. incognita (Mi), P. penetrans (Pp), and Mi + Pp inoculated simultaneously (MP) (n=10). To address the ambiguity of which PPN species infects the plant first, we included a sequential infection to see if prior infection of one species affects the other. For this, we added the treatments M. incognita with P. penetrans pre-infected plant (Mi after Pp, n=10) and P. penetrans with M. incognita pre-infected plant (Pp after Mi, n=10). One ten-day old B. nigra seedling was placed in the middle of the plate. In plates with pre-infection (Mi after Pp; Pp after Mi), the first nematode species was allowed to infect for 48 h, whereafter the seedling was transferred to a plate containing the nematode of interest. Nematodes touching the roots were counted after 4, 24, 48 and 72 h. After counting at 72 h, the roots were stained with acid-fuchsin (Bybd et al., 1983) to count the nematodes that have entered the roots. For single-species plates (Mi alone, Mi after Pp, Pp alone, and Pp after Mi), we divided the number of nematodes by 100. For the data from dual-species plates (Mi in MP and Pp in MP), we divided the number of nematodes by 50. M. incognita and P. penetrans have different feeding strategies and infection timelines. To facilitate uniformity in the context of concurrent infection, we defined early infection as the period from invasion until feeding site establishment for M. incognita, and for Pratylenchus spp. the period before complete invasion of the vascular tissues, including early penetration and migration, which can occur three hours to three weeks post-inoculation (Acedo and Rohde, 1971; Hol et al., 2016; van Dam et al., 2018). Statistical Analyses After data processing and compound annotation of the LC-MS data, the feature tables were exported to MetaboAnalyst 6.0 (Pang et al., 2024). The dataset was filtered based on their Interquartile Range. Given unequal replicate numbers and inherent variability among plants, Pareto-scaling was employed in addition to log 10 -transformation prior to the data analysis. The resulting dataset was analyzed using principal component analysis (PCA), followed by Permutation Multivariate Analysis of Variance (PERMANOVA). Differentially abundant (DA) features based on pairwise comparisons between the control and each of the nematode-treated plants (Mi, Pp, MP) were identified using the Volcano Plot function in MetaboAnalyst 6.0. The lists were exported to InteractiVenn, creating Venn diagrams (Heberle et al., 2015). We grouped features at superclass level via NPClassifier (Hyun Woo Kim et al., 2021) in SIRIUS/CANOPUS (Dührkop et al. , 2019, 2021) and assessed the difference in metabolite composition of nematode-treated roots using PERMANOVA via ‘adonis2’ function in R-package vegan, with Euclidean distance. The nematode attraction assay was analyzed using Friedman rank sum-test via the Friedman.test function in rstatix package (v4.1.2; R Core Team 2023) followed by post-hoc pairwise Wilcoxon test with Bonferroni correction . Differences in nematode root penetration, as well as in GSLs, GSL breakdown products and gene expression data, were identified using generalized linear model (GLM) with the lme4 package in R (Bates et al., 2015). Data were Log-transformed to attain normal distribution when needed. In datasets that do not follow a Gaussian distribution based on visual (histogram) and statistical ( Shapiro-Wilk Test ) assessments, specific GLM distribution functions were used depending on the characteristics of each dataset (Bates et al., 2015). Estimated marginal means (emmeans) in R was used for post-hoc comparison. Figures and plots were generated in R with ggplot2 or in MetaboAnalyst 6.0 and were optimized for publication in Inkscape 1.1.1 (3bf5ae0d25, 2021-09-20, inkscape.org). RESULTS Effects of Concurrent Nematode Infection on Root Metabolic Profiles Principal component analysis (PCA) showed that the root metabolic profile of MP-infected plants differed distinctly from that of control, Mi- and Pp-infected plants (Fig. 1A, Table S2). This concurs with the observation that the roots of MP-treated plants had the most differentially accumulated (DA) features, both for up (352)- and down (210)-regulated features (562 in total; Fig. 1b, e-f). The roots of Mi-treated plants had the least up- (28) or down- (24) regulated features (Fig. 1c, e-f), followed by Pp-treatment (77 down- and 77 up-regulated features; Fig. 1d, e-f). A complete list of DA features is given in Table S3-a-c. Some DA features could be annotated using our in-house library. Among these annotated features, we found that Pp treatment uniquely up-regulated sinigrin levels (Fig. S2; Table S3-b). MP treatment uniquely down-regulated desulfo-4-hydroxyglucobrassicin and desulfo-glucobrassicin, and uniquely up-regulated cis-12-oxo-phytodienoic acid (OPDA) (Fig. S2; Table S3-c). We used the features that could be assigned to superclass level (858 of 5919 features) to analyze differences in the root metabolic composition of control and nematode-treated plants. A PERMANOVA showed that nematode treatment significantly affected root chemical composition ( F = 1.941, df = 3, P = 0.004; Fig. 2, Table S4). The nematode treatments explained approximately 32.7% of the variation in the dataset. The grouped features could be divided into three clusters (Fig. 2). The first cluster (labelled I in Fig. 2) included compound groups that are different in one or two treatment groups (Fig. 2). For example, tyrosine alkaloids ( F = 5.268, df = 3, P = 0.023) were reduced in Mi- and MP-treated plants, but unchanged in Pp-infected plants. Nucleoside levels ( F = 3.608, df = 3, P = 0.05) were slightly increased in Pp-infected plants and slightly reduced in MP-treated plants, whereas polycyclic aromatic polyketides ( F = 4.731, df = 3, P = 0.028) were reduced in Mi-treated plants. The second cluster is characterized by reduced peak intensity in MP-treated plants (Fig. 2-II). Among these are defense-related compound classes, in the shikimate pathway, such as lignans ( F = 6.05, df = 3, P = 0.007) and phenylpropanoids ( F = 5.517, df = 3, P = 0.01). Similarly, aromatic polyketides ( F = 9.271, df = 3, P = 0.002), peptide alkaloids ( F = 11.528, df = 3, P = 0.001), anthranilic acid alkaloids ( F = 3.303, df = 3, P = 0.05), pseudoalkaloids ( F = 5.886, df = 3, P = 0.01), and amino acid glycosides ( F = 4.895, df = 3, P = 0.031) were also significantly reduced in response to MP treatment. The NPClassifer-based classification assigned many GSLs to the amino acid glycoside class (Table S3-d). The third cluster is composed of compound groups with increased peak intensities in response to MP treatment. This includes sesquiterpenoids ( F = 5.446, df = 3, P = 0.021), linear polyketides ( F = 4.653, df = 3, P = 0.024), fatty acyl glycosides ( F = 8.650, df = 3, P = 0.003), and octadecanoids ( F = 66.028, df = 3, P = 0.004). Moreover, MP treatment also uniquely accumulated eight features classified as octadecanoids (Fig. 2, fourth row) and uniquely reduced four features classified as lignans (Fig. 1F; Fig. S2, second and third row; Table S3-b). A complete list of PERMANOVA results can be found in Table S4. In addition, we analyzed the expression levels of common marker genes related to defense signaling pathways, such as PR1 , PAL1, and ERF1 . The jasmonate/ethylene regulator ERF1 was significantly down-regulated in all of the nematode-infected roots compared to the roots of control plants (GLM Gamma : χ 2 = 23.114, df = 3, P < 0.001; Fig. S3). With marginal statistical significance, the SA-responsive gene PR1 was down-regulated in MP-treated roots compared to untreated roots (GLM Gamma : χ 2 = 6.024, df = 3, P = 0.111, Fig. S3). PAL1 , however, was not differentially expressed by any of the nematode treatments (LM Gaussian : F = 0.76, df = 3, P = 0.540). Unfortunately, our analyses of the JA-related gene yielded low expression values and therefore could not be reliably interpreted. Altogether, these results suggest that metabolic changes in response to concurrent infection by M. incognita and P. penetrans differed from the changes in response to each PPN species alone. The differences are reflected in many defense-related compound classes such as lignans, phenylpropanoids, octadecanoids, and GSLs. Nematode-induced Changes in Root GSL and GSL Breakdown Products Based on the metabolic analyses, were we found the class amino acid glycosides, comprising many GSLs, significantly regulated, we performed targeted analyses of GSLs and their breakdown products. In addition, we analyzed the expression of GSL biosynthesis genes. The composition and relative abundance of GSL and breakdown products detected in the roots via targeted analysis is given in Table S5. In both targeted and untargeted analyses, we found that the levels of sinigrin were significantly higher in Pp-infected plants compared to control and MP-treated plants (GLM Gamma : χ 2 = 10.511, df = 3, P = 0.015, Fig. 3). In line with this observation, we found a significant increase of the degradation product, allyl ITC, in roots of Pp-treated plants compared to other treatments (LM Gaussian : F = 7.563, df = 3, P = 0.006; Fig. 3). The other breakdown products, 1-Cyano-2,3-epithiopropane (CETP) and allyl CN showed a similar pattern, though the differences were not statistically significant. Interestingly, the expression of CYP83A1 , involved in the synthesis of sinigrin, was significantly upregulated by Mi infection (LM Gaussian : F = 3.67, df = 3, P = 0.05; Fig. 3) compared to Pp- and MP-infections. For the indole GSLs, particularly for glucobrassicin (LM Gaussian : F = 5.098, df = 3, P = 0.017) and the neoglucobrassicin degradation product, 1-methoxyindole-3-acetonitrile (GLM Gamma : χ 2 = 12.129, df = 3, P = 0.007), we found that they are significantly reduced in the roots of MP-treated plants compared to other treatment groups (Fig. 4). Showing a similar pattern, although not statistically significantly so, were the precursor amino acid, tryptophan (LM Gaussian : F = 0.947, df = 3, P = 0.449), the GSLs downstream in the process, neoglucobrassicin (LM Gamma : χ 2 = 7.262, df = 3, P = 0.064), 4-hydroxyglucobrassicin (GLM Gamma : χ 2 = 1.674, df = 3, P = 0.643), and 4-methoxyglucobrassicin (GLM Gamma : χ 2 = 4.341, df = 3, P = 0.227), as well as the other indole GSL breakdown product, 4-methoxyindole-3-ACN (LM Gaussian : F = 2.305, df = 3, P = 0.129). Moreover, CYP79B2 was significantly upregulated by Mi-treatment (LM Gaussian : F = 3.74, df = 3, P = 0.049) compared to control and Pp-infected plants, while the transcription factor MYB122 was not differentially expressed by any of the nematode treatments (LM Gaussian : F = 2.27, df = 3, P = 0.143) (Fig. 4). Similar to the indole GSLs, the levels of the benzenic GSL gluconasturtiin (LM Gaussian : F = 22.128, df = 3, P < 0.001) and its degradation products, 2-phenylethyl ITC (LM Gaussian : F = 3.477, df = 3, P = 0.05) and 2-phenylethyl CN (LM: F = 4.753, df = 3, P = 0.021), had significantly lower levels in MP-treated plants (Fig. 5). Gluconasturtiin levels were significantly higher in Mi- (P < 0.001) and Pp-treated plants (P < 0.001) compared to MP-treated plants. In addition to its role in aliphatic GSL biosynthesis, the CYP83A1 gene is also involved in benzenic GSL biosynthesis. CYP83A1 is significantly downregulated in MP- and Pp-treated plants compared to Mi-treated plants (LM Gaussian : F = 3.67, df = 3, P = 0.05) (Fig. 5). The expression pattern of CYP83A1 was more similar to changes in this pathway than those in the aliphatic glucosinolate (sinigrin) pathways (Fig. 3). These results showed that single-species and concurrent nematode infections led to distinct changes in GSL and their breakdown products. In MP-treated plants, the levels of indole GSL and their breakdown products were reduced compared to Mi and PP plants. Both MP and Mi treatments altered the benzenic GSL, with marginally significant increased levels in response to Mi, yet significantly reduced levels in response to MP. Infection with Pp resulted in increased levels of sinigrin and its conversion products. Simultaneous Infection Affected the Early Performance of Each Nematode Species The attraction of M. incognita to uninfected roots differed from that of roots that were pre-infested or simultaneously infested with P. penetrans (Fig. 6a, Friedman χ 2 = 75.685, df = 4, P < 0.001; Fig. 6a). Over time, fewer M. incognita touched the roots when the plants were pre-inoculated with Pp, in particular, compared to MP (M in MP, Wilcoxon test: 4 h P = 0.018, 24 h P = 0.018, 48 h P = 0.006, 72 h P = 0.017; Fig. 6a). Similarly, fewer P. penetrans touched B. nigra roots when plants were pre-infested with Mi (Fig. 6b, Friedman χ 2 = 85.573, df = 4, P < 0.001), especially when compared to plants where P. penetrans was inoculated alone. By the end of the counting period, more M. incognita were touching the roots of MP plants (Fig. 6a), while more P. penetrans were touching the roots when inoculated alone (Fig. 6b). Inoculating Mi after or with Pp affected the penetration success of Mi at 3 dpi (GLM Gamma : χ 2 = 94.512, df = 2, P < 0.001; Fig. 6c). We found more M. incognita in roots of the MP-treated plants compared to Mi plants (EMMEANS: P < 0.001) or to roots pre-infected with P. penetrans (Mi after Pp) (EMMEANS: P < 0.001; Fig. 6c). Single and concurrent inoculations also differentially affected the P. penetrans penetration (GLM Gamma : χ 2 = 31.21, df = 2, P < 0.001; Fig. 6d). The numbers of P. penetrans found in the roots of dual-infected plants (P in MP) were significantly higher than in plants with prior M. incognita infection (Pp after Mi, EMMEANS: P = 0.001; Fig. 6d). There were also significantly more P. penetrans found in the roots of singly-infected plants (Pp alone) compared to Mi pre-infested plants (EMMEANS: P < 0.001; Fig. 6d). When both nematodes were inoculated at the same time (MP), we found significantly more M. incognita than P. penetrans in the roots (LM Gaussian : F = 4.5134, df = 1, P = 0.0477; Fig. 6e). Overall, these results showed that concurrent infection distinctly affects the early infection success of each nematode species, whereby the effects depended on the species and sequence of infection. DISCUSSION To understand how concurrent nematode infection affected the performance of each nematode species, we examined how infection by M. incognita and P. penetrans , alone and together, affected root metabolic profiles, particularly GSL and their breakdown products. Overall, concurrent infection led to a distinctly different root metabolic profile compared to that of single-species infections. This was mainly due to differences in defense-related compounds, such as lignans, phenylpropanoids, and GSLs. The different GSL classes responded individually to the different nematode treatments; Mi treatment increased benzenic GSLs, Pp treatment increased aliphatic GSLs, and MP treatment reduced indole GSLs. Furthermore, we found that M. incognita performs better when co-inoculated with P. penetrans , whereas P. penetrans performed worse when having to colonize a M. incognita infected plant. Concurrent PPN Infection Affected Root Metabolome Differently than Single-species Infection In natural environments, plants are often simultaneously attacked by multiple herbivores, resulting in complex interactions between signaling pathways (Bonnet et al., 2017 ; van Dam et al., 2018 ; Mbaluto et al., 2021 ). When herbivores with contrasting feeding strategies attack, the response to one herbivore can antagonize the response to another due to hormonal crosstalk or defense compound interaction (Pieterse et al., 2009 ; Bonnet et al., 2017 ; Mbaluto et al., 2021 ). For example, simultaneous attacks by aphids and caterpillars can weaken JA-mediated defenses, making plants more vulnerable to caterpillar damage (Soler et al., 2012 ; Ali and Agrawal, 2014 ). Our study examined the root metabolic changes in response to concurrent infection of two nematodes with contrasting feeding strategies – the sedentary endoparasite, Meloidogyne incognita and the migratory endoparasite, Pratylenchus penetrans. First, we hypothesized that each nematode species would elicit a specific root metabolic response. Our results showed that the overall root metabolic profiles of M. incognita and P. penetrans infected plants overall did not significantly differ from each other, nor did they differ from that of uninfected control plants (Fig. 1 a, Table S2 ). However, when looking into the differentially accumulated (DA) features in nematode-infected vs. control roots, we found several DA features responding to M. incognita and P. penetrans , with more DA compounds uniquely responding to each species, than DAs shared among these treatment groups (Fig. 1 e-f). Pratylenchus penetrans uniquely up-regulated sinigrin levels, which is a typical response to tissue damage in Brassica spp. (Traw, 2002 ). The continuous tissue damage caused by the migratory P. penetrans may have caused the increased level of the toxic allyl ITC. This response is less expected of M. incognita infections, as early Meloidogyne spp. infection typically causes less tissue damage (Gheysen and Mitchum, 2011 ; van Dam et al., 2018 ). Moreover, in the infection process, Meloidogyne spp. secrete effectors to down-regulate plant defense responses (Gheysen and Mitchum, 2011 ; Kyndt et al., 2012a ). We further hypothesized that concurrent infections of M. incognita and P. penetrans attenuate changes in the root metabolic profile. Concurrent infection resulted in a distinct root metabolic profile (Fig. 1 a). Other than expected, double infected plants showed the highest numbers of significantly upregulated and downregulated DA features (Fig. 1 b), indicating a more complex and intense metabolic response. Interestingly, the distinct changes in root metabolic profiles could be predominantly attributed to the differential accumulation of several defense compounds in response to MP treatment (Fig. 2 ). This may suggest that dual infection may have amplified the plant's defense responses, resulting in the simultaneous regulation of multiple defense-related pathways. The distinguished increase of OPDA levels and those of other octadecanoids during dual infection suggests that the octadecanoid pathway may have been involved in enhancing the production of defensive metabolites (León and Sánchez-Serrano, 1999 ; Papazian et al., 2019 ). On the other hand, dual infection also reduced the levels of lignans and phenylpropanoids, two distinct groups of compounds with critical roles in plant defense (Dixon et al., 2002 ; Ražná et al., 2021 ). Both compound classes have been implicated in plant responses to the RKN M. javanica in tomatoes, as demonstrated by the upregulation of transcripts encoding for enzymes involved in their biosynthesis (Kamali et al., 2022 ). Compounds classified as alkaloids, i.e. , peptide alkaloids, aromatic alkaloids, anthranilic acid alkaloids, as well as aromatic polyketides were also reduced in MP-treated roots. The reduction of these compounds suggests a more compromised plant defense under double infection. Moreover, amino acid glycosides, a class in which several GSLs are found, were also significantly reduced in response to MP treatment. GSLs are key defense compounds in Brassica plants and are known for their defensive role against herbivores, including nematodes (Potter et al., 2000 ). This motivated thus to perform targeted analysis of GSLs and GSL breakdown products to further understand their role in the response to dual and single-species nematode infections. Nematode Infection Affects Specific GSL Metabolic Pathways Our results demonstrated that single-species and concurrent infections by M. incognita and P. penetrans led to distinct alterations in the root GSL profile of B. nigra . The ‘mustard oil bomb’ mechanism, where tissue damage triggers the GSL hydrolysis by the enzyme myrosinase (Abdel-Massih et al., 2023 ), produces toxic breakdown products that can affect insects, microbes, and nematodes (van Dam et al., 2009 ; Eugui et al., 2022 ; Sontowski et al., 2022 ; Chekanai et al., 2024 ). The levels of sinigrin, as well as its breakdown product, allyl ITC, were induced by P. penetrans (Fig. 2 ). This suggests an induced response that may be associated with the direct and extensive mechanical damage inflicted by P. penetrans on root tissues. Allyl ITC, in particular, was shown to reduce the motility and increase the mortality of P. penetrans in a time- and dose-dependent manner (Chekanai et al., 2024 ). In MP-infected plants, however, sinigrin and allyl ITC levels were significantly reduced compared to that of Pp-treated plants. This reduction suggests that the presence of M. incognita may have interfered with the sinigrin-mediated response of B. nigra towards P. penetrans. Follow-up bioassays using mutants impaired in the production of sinigrin or allyl-ITC in their roots, could further elucidate the role of sinigrin and its breakdown products in the defense against P. penetrans. The benzenic GSL, gluconasturtiin and its degradation products 2-phenylethyl ITC and 2-phenylethyl CN were also reduced in MP-treated plants, whereas these compounds were slightly higher in Mi-treated plants (Fig. 5 ). Indeed, gluconasturtiin and 2-phenylethyl ITC are among the GSLs deemed effective in suppressing nematode populations (Potter et al., 2000 ; Eugui et al., 2022 ), in particular those of Pratylenchus spp. (Potter et al., 1998 ; Potter et al., 2000 ). Our results thus align with previous reports that gluconasturtiin and 2-phenylethyl ITC play a role in mediating plant responses to nematode infections (Potter et al., 2000 ; Eugui et al., 2022 ). Concurrent infection reduced the less abundant indole GSLs and their breakdown products, particularly glucobrassicin and 1-methoxyindole-ACN (Fig. 4 ). Root-knot nematode feeding leads to significant changes in plant root tissue, starting with the establishment of feeding sites that result in the formation of giant cells, creating root galls (Gheysen and Mitchum, 2011 ; Mbaluto et al., 2021 ). Transcriptomic analysis of giant cells obtained via laser dissection has shown that the indole GSL biosynthesis gene, CYP79B2 was down-regulated in the giant cells but up-regulated in surrounding vascular tissues (Barcala et al., 2010 ). This shows that the metabolic route involving indole GSL can be among the defense pathways responding to RKN infection and may be manipulated by the nematode for successful feeding. Our results point in a similar direction. We also found a significant upregulation of CYP79B2 in M. incognita- infected roots (Fig. 4 ). Indole GSLs, in particular, are known for their anti-pathogen effects (Clay et al., 2009 ). Increased indole GSL levels have also been correlated with response to aphid feeding (Kim and Jander, 2007 ; Pajar et al., 2024 ). Aphids and nematodes, particularly, RKN, share similarities regarding their interactions with host plants. Both organisms use effectors to manipulate plant metabolic processes while creating their respective feeding sinks (Gheysen and Mitchum, 2011 ; Züst and Agrawal, 2016 ). Given this similarity, it is not surprising that indole GSL could be one of the metabolites that respond to M. incognita infection. If indole GSLs indeed confer resistance to RKN, then the reduction of glucobrassicin and 1-methoxyindole-3-ACN in response to the MP treatment may have potentially benefited M. incognita , which correlates to better early infection performance compared to single-species inoculation. However, further evaluation of the specific effects of indole GSL and the breakdown products is needed to assess their defensive role against nematodes. Our results also showed that even the least abundant GSL breakdown products responded to nematode infection. In particular, we found a significant reduction of the less abundant 1-methoxyindole-3-ACN (Fig. 4 ) and 2-phenylethyl CN (Fig. 5 ) in the roots with MP treatment compared to Mi-treated and control plants. Due to their high GSL concentrations, several Brassica species are effective biofumigants (Dahlin and Hallmann, 2020 ; Eugui et al., 2022 ). Biofumigation incorporates chopped plant materials into the soil, releasing toxic GSL breakdown products with nematocidal effects. This process significantly reduced nematode populations (Potter et al., 1998 ; Eugui et al., 2022 ), including over 60% reduction in galls and egg masses of Meloidogyne spp. in greenhouse settings (Oliveira et al., 2011 ; Curto et al., 2016 ). While studies on the effect of GSL breakdown products mostly focused on the more abundant and biologically active ITCs, these results indicate that also nitriles/CNs may play a role in plant-PPN interactions. Insights on the Relationship of M. incognita and P. penetrans in Concurrent Infection We also considered the presence of nematodes in close proximity to the roots (touching the root, attraction) and inside the roots (penetration) as measures of early infection success. Our results suggest that M. incognita is more successful when co-inoculated with P. penetrans , while P. penetrans is unaffected by co-inoculation but was more successful in the absence of prior M. incognita infection (Fig. 6 ). This implies that concurrent PPN infection affects each nematode species differently. Meloidogyne spp. and Pratylenchus spp. were reported to behave antagonistically in several plant pathosystems (Gay and Bird, 1973 ; Chapman and Turner, 1975 ; Fontana et al., 2015 ), but there are not many studies showing the possible mechanisms for this antagonism, particularly in relation to plant-mediated responses. We found that MP treatment, among other changes in root secondary metabolite, reduced indole (Fig. 4 ) and benzenic (Fig. 5 ) GSLs, as well as their breakdown products. The reduction of specific GSL and their breakdown products, as well as the down-regulation of metabolites belonging to other defense-related classes, such as phenylpropanoids and lignans (Fig. S2 , Table S4), suggest that the plant defenses are compromised under dual infection. The enhanced OPDA and other octadecanoids (Fig. S2 ) levels in MP-treated plants suggest increased signaling in the JA pathway, which in turn may have suppressed the SA signaling pathway. Since M. incognita is more susceptible to SA-based defenses (Bonnet et al., 2017 ; Gheysen and Mitchum, 2019 ), the metabolic changes in response to concurrent infection may have favored M. incognita. This is associated with the greater early infection success of M. incognita in concurrent infections compared to single-species infection (Fig. 6 ). This is, however, not the case for P. penetrans , whose early infection success did not significantly differ when it was infected alone or with M. incognita. We observed an overall decline in the number of nematodes in contact with the roots over time, except in the Pp and Pp in MP treatment groups. The decline is likely due to the fact that M. incognita J2 rapidly penetrate the roots and thus are not visible outside anymore. To confirm this, we stained the roots after counting at 72h. The stained roots of MP-treated plants showed that more M. incognita were able to enter the roots at 3 dpi compared to P. penetrans (Fig. 5 ). According to Mateille et al. ( 2020 ), PPN species may affect each other via competition when both species use the same limited resources. It is thus possible that, whereas the attenuated defense response favored M. incognita in MP treatments, M. incognita may have also outcompeted P. penetrans in terms of limited resources, such as nutrients and infection sites. Though we did not include root metabolic measurements of the Pp-after-Mi treatment, the results of the nematode performance assay showed that P. penetrans is negatively affected by prior M. incognita infection. Nevertheless, more bioassays, particularly using mutants impaired in previously mentioned signaling pathways, are needed to elucidate the underlying mechanism of nematode-nematode interactions in concurrent infection. Taken together, we showed that the root metabolic profile of dual nematode-infected B. nigra is markedly different from those of plants infected by either M. incognita or P. penetrans alone. This suggests that the concurrent infection by both nematodes triggers a unique metabolic response that is not merely a combination of the individual responses to each nematode. This novel information generated from untargeted metabolic analysis could serve as basis for generating further hypotheses in exploring specific plant responses to single and dual nematode infections. In particular GSL and their breakdown products responded differently to the nematode treatments. Considering the specificity of the GSL response, we suggest to further explore the specificity of GSL and GSL breakdown products as defenses against different plant parasitic nematode species. This knowledge can potentially augment the effectiveness of biofumigation and other related crop protection strategies. Declarations Funding We acknowledge support by the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig (funded by the German Research Foundation (DFG), grant no. FZT 118, 202548816), the International Max Planck Research School "Chemical Communication in Ecological Systems" and Leibniz Institute for Vegetable and Ornamental Crops (IGZ) for the funding to JAP and NMvD. We acknowledge the Erasmus + mobility fund - KA1 for the Erasmus Mundus Joint Master Degrees Programme of the European Commission under the PLANT HEALTH Project for providing support to PO, as well as the Erasmus + mobility fund and VLIR-OUS for the Masters in Agro- and Environmental Nematology program that supported AL. Competing interests The authors declare no conflict of interest Author Contribution JAP and NMvD conceptualized the study and designed the experiments. JAP and PO performed the greenhouse experiment, processed the root samples, and performed glucosinolate quantification. JAP and ALL optimized and performed the nematode performance assays. JAP and SD performed untargeted metabolomic analysis. JAP and FSH acquired and analyzed the data for glucosinolate breakdown products. JAP performed the gene expression analysis. JAP wrote the first draft of the paper with support from NMvD. All authors contributed to the final manuscript. Acknowledgement We would like to thank Dr. Rebekka Sontowski, Dr. Axel Touw, Maria Skoruppa, and Jessica Eichhorn for their assistance during the GSL and GSL breakdown product analyses. We also want to thank Prof. Nicole Viaene (Department of Plant Sciences, Flanders Institute for Agriculture, Fisheries and Food, Belgium) and Patricia Poot (Bejo BV, Warmenhuizen, The Netherlands) for providing us with nematodes. Data Availability Data supporting this study are included in the manuscript and supplementary files. References Abdel-Massih RM, Debs E, Othman L, Attieh J, Cabrerizo FM (2023) Glucosinolates, a natural chemical arsenal: More to tell than the myrosinase story. Front Microbiol 14: 1–15 Acedo JR, Rohde RA (1971) Histochemical Root Pathology of Brassica oleracea capitata L. Infected by Pratylenchus penetrans (Cobb) Filipjev and Schuurmans Stekhoyen (Nematoda: Tylenchidae). J Nematol 3: 62–68 Agerbirk N, Olsen CE (2012) Glucosinolate structures in evolution. Phytochemistry 77: 16–45 Ali JG, Agrawal AA (2014) Asymmetry of plant-mediated interactions between specialist aphids and caterpillars on two milkweeds. Funct Ecol 28: 1404–1412 Barcala M, García A, Cabrera J, Casson S, Lindsey K, Favery B, García-Casado G, Solano R, Fenoll C, Escobar C (2010) Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells. Plant J 61: 698–712 Bartlem DG, Jones MGK, Hammes UZ (2014) Vascularization and nutrient delivery at root-knot nematode feeding sites in host roots. J Exp Bot 65: 1789–1798 Bates D, Mächler, MartinBates D, Mächler M, Bolker B, Walker S. 2015. Fitting Linear Mixed-Effects Models Using lme4. Journal of Statistical Software 67 1–48., Bolker B, Walker S (2015) Fitting Linear Mixed-Effects Models Using lme4. J Stat Softw 67: 1–48 Bonnet C, Lassueur S, Ponzio C, Gols R, Dicke M, Reymond P (2017) Combined biotic stresses trigger similar transcriptomic responses but contrasting resistance against a chewing herbivore in Brassica nigra . BMC Plant Biol 17: 1–14 Bybd DW, Kirkpatrick T, Barker KR (1983) An improved technique for clearing and staining plant tissues for detection of nematodes. J Nematol 15: 142–143 Chapman RA, Turner DR (1975) Effect of Meloidogyne incognita on Reproduction of Pratylenchus penetrans in Red Clover and Alfalfa. J Nematol 7: 6–9 Chekanai V, Neilson R, Roberts D, Edwards S, Back M (2024) In vitro nematicidal efficacy of brassica-derived isothiocyanates against the root lesion nematode, Pratylenchus penetrans . Nematology 26: 899–908 Clay NK, Adio AM, Denoux C, Jander G, Ausubel FM (2009) Glucosinolate Metabolites Required for an Arabidopsis Innate Immune Response. Science (80- ) 323: 95–101 Curto G, Dallavalle E, Matteo R, Lazzeri L (2016) Biofumigant effect of new defatted seed meals against the southern root-knot nematode, Meloidogyne incognita . Ann Appl Biol 169: 17–26 Dahlin P, Hallmann J (2020) New Insights on the Role of Allyl Isothiocyanate in Controlling the Root Knot Nematode Meloidogyne ha P la . Plants. doi: 10.3390/plants9050603 van Dam NM, Tytgat TOG, Kirkegaard JA (2009) Root and shoot glucosinolates: a comparison of their diversity, function and interactions in natural and managed ecosystems. Phytochem Rev 8: 171–186 van Dam NM, Witjes L, Svatoš A (2003) Interactions between aboveground and belowground induction of glucosinolates in two wild Brassica species. New Phytol 161: 801–810 van Dam NM, Wondafrash M, Mathur V, Tytgat TOG (2018) Differences in hormonal signaling triggered by two root-feeding nematode species result in contrasting effects on aphid population growth. Front Ecol Evol. doi: 10.3389/fevo.2018.00088 Desmedt W, Mangelinckx S, Kyndt T, Vanholme B (2020) A Phytochemical Perspective on Plant Defense Against Nematodes. Front Plant Sci. doi: 10.3389/fpls.2020.602079 Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MSS, Wang L (2002) The phenylpropanoid pathway and plant defence - A genomics perspective. Mol Plant Pathol 3: 371–390 Dührkop K, Fleischauer M, Ludwig M, Aksenov AA, Melnik A V, Meusel M, Dorrestein PC, Rousu J, Böcker S (2019) Brief CommuniCation SIRIUS 4: a rapid tool for turning tandem mass spectra into metabolite structure information. Nat Methods 16: 299–302 Dührkop K, Nothias L-F, Fleischauer M, Reher R, Ludwig M, Hoffmann MA, Petras D, Gerwick WH, Rousu J, Dorrestein PC, et al (2021) Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra. Nat Biotechnol 39: 462–471 Eloh K, Sasanelli N, Maxia A, Caboni P (2016) Untargeted Metabolomics of Tomato Plants after Root-Knot Nematode Infestation. J Agric Food Chem 64: 5963–5968 Eugui D, Escobar C, Velasco P, Poveda J (2022) Glucosinolates as an effective tool in plant-parasitic nematodes control: Exploiting natural plant defenses. Appl Soil Ecol 176: 104497 Fontana LF, Dias-Arieira CR, Mattei D, Severino JJ, Biela F, Arieira JDO (2015) Competition between Pratylenchus zeae and Meloidogyne incognita on sugarcane. Nematropica 45: 1–8 Fosu-Nyarko J, Jones MGK (2016) Advances in Understanding the Molecular Mechanisms of Root Lesion Nematode Host Interactions. doi: 10.1146/annurev-phyto-080615-100257 Gay CM, Bird GW (1973) Influence of Concomitant Pratylenchus brachyurus and Meloidogyne spp. on Root Penetration and Population Dynamics. J Nematol 5: 212–7 Gheysen G, Mitchum MG (2011) How nematodes manipulate plant development pathways for infection. Curr Opin Plant Biol 14: 415–421 Gheysen G, Mitchum MG (2019) Phytoparasitic nematode control of plant hormone pathways. Plant Physiol 179: 1212–1226 Grosser K, van Dam NM (2017) A straightforward method for glucosinolate extraction and analysis with high-pressure liquid chromatography (HPLC). JoVE (Journal Vis Exp 121: e55425 Hanschen FS (2024) Acidification and tissue disruption affect glucosinolate and S-methyl-l-cysteine sulfoxide hydrolysis and formation of amines, isothiocyanates and other organosulfur compounds in red cabbage ( Brassica oleracea var. capitata f. rubra ). Food Res Int 178: 114004 Hanschen FS, Schreiner M (2017) Isothiocyanates, nitriles, and epithionitriles from glucosinolates are affected by genotype and developmental stage in Brassica oleracea varieties. Front Plant Sci 8: 1–17 Heberle H, Meirelles GV, Da Silva FR, Telles GP, Minghim R (2015) InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics 16: 169 Hofmann J, El A, Ashry N El, Anwar S, Erban A, Kopka J, Grundler F (2010) Metabolic profiling reveals local and systemic responses of host plants to nematode parasitism. Plant J 62: 1058–1071 Hol WHG, Raaijmakers CE, Mons I, Meyer KM, van Dam NM (2016) Root-lesion nematodes suppress cabbage aphid population development by reducing aphid daily reproduction. Front Plant Sci 7: 1–11 Hooper DJ, Hallmann J, Subbotin SA (2005) Methods for extraction, processing and detection of plant and soil nematodes. Plant Parasit. nematodes Subtrop. Trop. Agric. Wallingford UK: CABI Publishing, pp 53–86 Hyun Woo Kim, Mingxun Wang, Christopher A. Leber, Louis-Félix Nothias, Raphael Reher, Kyo Bin Kang, Justin J. J. van der Hooft, Pieter C. Dorrestein, William H. Gerwick, Garrison W. Cottrell (2021) NPClassifier: A Deep Neural Network-Based Structural Classification Tool for Natural Products | Enhanced Reader. J Nat Prod 2795–2807 Jones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MGK, Kikuchi T, Manzanilla-López R, Palomares-Rius JE, Wesemael WML, et al (2013) Top 10 plant-parasitic nematodes in molecular plant pathology. Mol Plant Pathol 14: 946–961 Kamali S, Javadmanesh A, Stelinski LL, Kyndt T, Seifi A, Cheniany M, Zaki-Aghl M, Hosseini M, Heydarpour M, Asili J, et al (2022) Beneficial worm allies warn plants of parasite attack below-ground and reduce above-ground herbivore preference and performance. Mol Ecol 31: 691–712 Kantor M, Handoo Z, Kantor C, Carta L (2022) Top Ten Most Important U.S.-Regulated and Emerging Plant-Parasitic Nematodes. Hortic Rev. doi: 10.3390/horticulturae Kim JH, Jander G (2007) Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate. Plant J 49: 1008–1019 Kliebenstein DJ, Gershenzon J, Mitchell-Olds T (2001) Comparative Quantitative Trait Loci Mapping of Aliphatic, Indolic and Benzylic Glucosinolate Production in Arabidopsis thaliana Leaves and Seeds. Genetics 159: 359–370 Kyndt T, Denil S, Haegeman A, Trooskens G, Bauters L, Van Criekinge W, De Meyer T, Gheysen G (2012a) Transcriptional reprogramming by root knot and migratory nematode infection in rice. New Phytol 196: 887–900 Kyndt T, Nahar K, Haegeman A, De Vleesschauwer D, Höfte M, Gheysen G (2012b) Comparing systemic defence-related gene expression changes upon migratory and sedentary nematode attack in rice. Plant Biol 14: 73–82 Kyndt T, Vieira P, Gheysen G, de Almeida-Engler J (2013) Nematode feeding sites: Unique organs in plant roots. Planta 238: 807–818 Lancashire P, Bleiholde H, van Den Boom T, Langeluddeke P, Stauss R, Weber E, Witzenberger A. (1991) A uniform decimal code for growth stages of crops and weeds. Ann Appl Biol 119: 561-601. León J, Sánchez-Serrano JJ (1999) Molecular biology of jasmonic acid biosynthesis in plants. Plant Physiol Biochem 37: 373–380 Liébanas G, Castillo P (2004) Host suitability of some crucifers for root-knot nematodes in southern Spain. Nematology 6: 125–128 Livak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25: 402–408 Lohmann M, Scheu S, Müller C (2009) Decomposers and root feeders interactively affect plant defence in Sinapis alba. Oecologia 160: 289–298 Mateille T, Tavoillot J, Goillon C, Pares L, Lefèvre A, Védie H, Navarrete M, Palloix A, Sage-Palloix A-M, Castagnone-Sereno P, et al (2020) Competitive interactions in plant-parasitic nematode communities affecting organic vegetable cropping systems. Crop Prot 135: 105206 Mbaluto CM, Ahmad EM, Fu M, Martínez-Medina A, van Dam NM (2020) The impact of Spodo p tera exigua herbivory on Meloidogyne incognita -induced root responses depends on the nematodes’ life cycle stages. AoB Plants 12: plaa029 Mbaluto CM, Vergara F, van Dam NM, Martínez-Medina A (2021) Root infection by the nematode Meloidogyne incognita modulates leaf antiherbivore defenses and plant resistance to Spodoptera exigua . J Exp Bot 72: 7909–7926 Mitchum MG, Rocha RO, Huang G, Maier TR, Baum TJ, Hussey RS (2023) Genome-Guided Reanalysis of Root-Knot Nematode Meloidogyne incognita Esophageal Gland Cell-Enriched Sequence Tag Libraries: A Resource for the Discovery of Novel Effectors. PhytoFrontiers. doi: 10.1094/PHYTOFR-09-22-0099-A Mukhopadhyay & Roy, K. AK (2006) Community analysis of major plant parasitic nematodes associated with vegetable crops in Eastern and Northeastern India. Int J Nematol 16: 194--199. Nicol JM, Turner SJ, Coyne DL, Nijs L Den, Hockland S (2011) Current nematode threats to world agriculture. Genomics Mol. Genet. Plant-Nematode Interact. pp 21–43 Oliveira RDL, Dhingra OD, Lima AO, Jham GN, Berhow MA, Holloway RK, Vaughn SF (2011) Glucosinolate content and nematicidal activity of Brazilian wild mustard tissues against Meloidogyne incognita in tomato. Plant Soil 341: 155–164 Oñate-Sánchez L, Vicente-Carbajosa J (2008) DNA-free RNA isolation protocols for Arabidopsis thaliana , including seeds and siliques. BMC Res Notes 1: 93 Pajar JA, Otto P, Leonar AL, Döll S, van Dam NM (2024) Dual nematode infection in Brassica nigra affects shoot metabolome and aphid survival in distinct contrast to single-species infection. Journal of Experimental Botany, Volume 75, 22:7317–7336 Pang Z, Lu Y, Zhou G, Hui F, Xu L, Viau C, Spigelman AF, MacDonald PE, Wishart DS, Li S, et al (2024) MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res 52: W398–W406 Papazian S, Girdwood T, Wessels BA, Poelman EH, Dicke M, Moritz T, Albrectsen BR (2019) Leaf metabolic signatures induced by real and simulated herbivory in black mustard ( Brassica nigra ). Metabolomics 15: 1–16 Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5: 308–316 Potter MJ, Davies K, Rathjen AJ (1998) Suppressive impact of glucosinolates in Brassica vegetative tissues on root lesion nematode Pratylenchus neglectus. J Chem Ecol 24: 67–80 Potter MJ, Vanstone VA, Davies KA, Rathjen AJ (2000) Breeding to increase the concentration of 2-phenylethyl glucosinolate in the roots of Brassica napus . J Chem Ecol 26: 1811–1820 Ražná K, Nôžková J, Vargaová A, Harenčár Á, Bjelková M (2021) Biological functions of lignans in plants. Agric 67: 155–165 Ren Z, Li Y, Fang W, Yan D, Huang B, Zhu J, Wang X, Wang X, Wang Q, Guo M, et al (2018) Evaluation of allyl isothiocyanate as a soil fumigant against soil-borne diseases in commercial tomato ( Lycopersicon esculentum Mill.) production in China. Pest Manag Sci 74: 2146–2155 Rutter WB, Franco J, Gleason C (2022) Rooting Out the Mechanisms of Root-Knot Nematode-Plant Interactions. Annu Rev Phytopathol 60: 43–76 Sijmons PC, Atkinson H, Wyss U (1994) Parasitic Srategies of Root Nematodes and Associated Host Cell Responses. Annu Rev Phytopathol 32: 235–59 Soler R, Badenes-Pérez FR, Broekgaarden C, Zheng SJ, David A, Boland W, Dicke M (2012) Plant-mediated facilitation between a leaf-feeding and a phloem-feeding insect in a brassicaceous plant: From insect performance to gene transcription. Funct Ecol 26: 156–166 Sontowski R, Guyomar C, Poeschl Y, Weinhold A, van Dam NM, Vassão DG (2022) Mechanisms of Isothiocyanate Detoxification in Larvae of Two Belowground Herbivores, Delia radicum and D. floralis (Diptera: Anthomyiidae). Front Physiol 13: 1–17 Tileubayeva Z, Avdeenko A, Avdeenko S, Stroiteleva N, Kondrashev S (2021) Plant-parasitic nematodes affecting vegetable crops in greenhouses. Saudi J Biol Sci 28: 5428–5433 Touw AJ, Mogena AV, Maedicke A, Sontowski R, van Dam NM, Tsunoda T (2020) Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in Brassica rapa . Front Plant Sci 10: 1653 Traw MB (2002) Is induction response negatively correlated with constitutive resistance in black mustard? Evolution (N Y) 56: 2196–2205 Tsunoda T, Krosse S, van Dam NM (2017) Root and shoot glucosinolate allocation patterns follow optimal defence allocation theory. J Ecol 105: 1256–1266 Vieira P, Maier TR, Akker SEDEN, Howe DK, Zasada I, Baum TJ, Eisenback JD, Kamo K (2018) Identification of candidate effector genes of Pratylenchus penetrans . 9: 1887–1907 Wang C, Lower S, Thomas VP, Williamson VM (2010) Root-Knot Nematodes Exhibit Strain-Specific Clumping Behavior That Is Inherited as a Simple Genetic Trait. PLoS One 5: e15148 Wang C, Lower S, Williamson VM (2009) Application of Pluronic gel to the study of root-knot nematode behaviour. Nematology 11: 453–464 Yu J, Vallad GE, Boyd NS (2019) Evaluation of allyl isothiocyanate as a soil fumigant for tomato ( Lycopersicon esculentum Mill.) production. Plant Dis 103: 2764–2770 Zhang X, Song M, Gao L, Tian Y (2023) Metabolic variations in root tissues and rhizosphere soils of weak host plants potently lead to distinct host status and chemotaxis regulation of Meloidogyne incognita in intercropping. Mol Plant Pathol 1–16 Züst T, Agrawal AA (2016) Mechanisms and evolution of plant resistance to aphids. Nat Plants 2: 15206 Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.xlsx GelRNAextraction.png Cite Share Download PDF Status: Published Journal Publication published 10 Sep, 2025 Read the published version in Journal of Chemical Ecology → Version 1 posted Editorial decision: Revision requested 08 Jul, 2025 Reviews received at journal 07 Jul, 2025 Reviews received at journal 03 Jul, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers agreed at journal 14 Jun, 2025 Reviewers invited by journal 14 Jun, 2025 Editor assigned by journal 14 Jun, 2025 Submission checks completed at journal 11 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6835102","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482528545,"identity":"073438e7-c246-4970-b316-9f7c098f7a5f","order_by":0,"name":"Jessil Ann Pajar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACxgYGBgMGBhseZAGitKSRoAUKDqOYgR8wz25+UPBzz3kZ/vYG1s0Fvxhk+wk6bM4xA8OeZ7d5JM4cYLs9s4/BeCYhaxhnJBgY8By4zWMgkcB2m7eHIXHDAYJa0j8Y/jlwjsdA/gFEy37CWnIMjHkOHADawsB2m+cH0BbCfjlTYCxzIBnol8S22zMbJIxnELLFcHb7NsM3B+zs+dsPH7td8MdGtr+BkJYZDGwGUAsbmBnbJAg5i4FBXoKB+QGMw8zwh7COUTAKRsEoGHkAAIqmRA+v7Vh6AAAAAElFTkSuQmCC","orcid":"","institution":"Max Planck Institute for Chemical Ecology","correspondingAuthor":true,"prefix":"","firstName":"Jessil","middleName":"Ann","lastName":"Pajar","suffix":""},{"id":482528546,"identity":"458e66bf-b4bc-475f-8247-f6f2344c0624","order_by":1,"name":"April Lyn Leonar","email":"","orcid":"","institution":"German Centre for Integrative Biodiversity Research (iDiv)","correspondingAuthor":false,"prefix":"","firstName":"April","middleName":"Lyn","lastName":"Leonar","suffix":""},{"id":482528548,"identity":"4b75298e-b0a9-4b63-8797-0cf1863d2261","order_by":2,"name":"Pius Otto","email":"","orcid":"","institution":"German Centre for Integrative Biodiversity Research (iDiv)","correspondingAuthor":false,"prefix":"","firstName":"Pius","middleName":"","lastName":"Otto","suffix":""},{"id":482528550,"identity":"3b361430-80f7-478f-b51b-65c4d27889b8","order_by":3,"name":"Franziska Sabine Hanschen","email":"","orcid":"","institution":"Leibniz Institute for Vegetable and Ornamental Crops (IGZ) e.V","correspondingAuthor":false,"prefix":"","firstName":"Franziska","middleName":"Sabine","lastName":"Hanschen","suffix":""},{"id":482528551,"identity":"9d7ae610-567b-40bd-81b4-914ac3753991","order_by":4,"name":"Stefanie Döll","email":"","orcid":"","institution":"German Centre for Integrative Biodiversity Research (iDiv)","correspondingAuthor":false,"prefix":"","firstName":"Stefanie","middleName":"","lastName":"Döll","suffix":""},{"id":482528552,"identity":"78e17208-900f-4de4-861b-97249b4fbceb","order_by":5,"name":"Nicole M. Dam","email":"","orcid":"","institution":"Leibniz Institute for Vegetable and Ornamental Crops (IGZ) e.V","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"M.","lastName":"Dam","suffix":""}],"badges":[],"createdAt":"2025-06-06 08:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6835102/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6835102/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10886-025-01637-8","type":"published","date":"2025-09-10T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86626649,"identity":"add3fac2-72e7-4289-bce8-21d627c3d311","added_by":"auto","created_at":"2025-07-14 05:30:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":141907,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic profile of \u003cem\u003eBrassica nigra\u003c/em\u003e roots (a-f), ten days after single (Mi, Pp) or dual (MP) nematode treatments. Metabolic profiles were constructed based on LC-qToF-MS/MS analyses in positive ionization mode. (a) Separation of root metabolic profiles of nematode-treated plants and mock-inoculated control plants shown in 2D scores plot following \u003cem\u003ePrincipal Component Analysis\u003c/em\u003e (PCA). Points represent replicates per treatment group (n\u003csub\u003econtrol, Mi \u003c/sub\u003e= 5; n\u003csub\u003ePp, MP\u003c/sub\u003e = 3), percentages at the axes indicate the variation explained by each component, ellipses indicate 95% confidence interval. Volcano plots (b, c, d) showing differentially abundant metabolites in the roots of (b) MP-, (c) Mi-, and (d) Pp-infested versus control plants. Metabolites were considered differentially abundant (DA) based on the following thresholds: fold change \u0026gt;2.0, P ≤ 0.05 (t-test). The colored points represent metabolite groups classified via NPClassifier. The number of unique and shared DA metabolites are summarized in Venn Diagrams (e-f). These diagrams show the number of down- (e) and up- (f) regulated metabolites in the roots. The complete list of DA metabolites is given in Table S3-a-c. Abbreviations: Mi= \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, Pp= \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, MP= \u003cem\u003eM. incognita\u003c/em\u003e + \u003cem\u003eP. penetrans\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/4bac23bf4fc5440ec7a5a5fb.png"},{"id":86626664,"identity":"4da3d024-100d-4a23-ac4f-9b023ae8e1ea","added_by":"auto","created_at":"2025-07-14 05:30:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154839,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap showing clustering of features grouped according to NPClassifier superclass from untargeted secondary metabolite analysis of \u003cem\u003eBrassica nigra\u003c/em\u003e roots from plants infected with single (Mi, Pp) and dual nematode (MP) species, as well as uninfected (control) plants. Clustering was based on \u003cem\u003eEuclidean distance\u003c/em\u003e and \u003cem\u003eWard algorithm\u003c/em\u003e methods. Dark blue and yellow gradient represent the relative abundance of compounds based on log\u003csub\u003e10\u003c/sub\u003e-transformed and Pareto-scaled peak intensities, shown as average per treatment (n=3-5). Dark blue indicates higher relative intensity; yellow indicates lower relative intensity. Compound groups written in bold letters are significantly different at P\u0026lt;0.05. Abbreviations: Mi= \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, Pp= \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, MP= \u003cem\u003eM. incognita\u003c/em\u003e + \u003cem\u003eP. penetrans\u003c/em\u003e\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/3c3a27e57f40d2739ffa7124.png"},{"id":86626651,"identity":"66960264-4950-4834-938b-3ae6879d40e2","added_by":"auto","created_at":"2025-07-14 05:30:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106284,"visible":true,"origin":"","legend":"\u003cp\u003eContents of the aliphatic glucosinolate sinigrin (allyl glucosinolate) and its breakdown products in \u003cem\u003eBrassica nigra\u003c/em\u003e roots, including the relative expression of CYP83A1 gene as they occur in the aliphatic glucosinolate metabolism pathway. Boxes and names in gray are part of the pathways that were not measured in the experiment. Compounds below the broken line are breakdown products. The biological replicates per treatment (n\u003csub\u003econtrol, Mi \u003c/sub\u003e= 5; n\u003csub\u003ePp, MP\u003c/sub\u003e = 3) are represented by points in the boxplots. Boxplots labeled with different letters are significantly different as per the estimated marginal means (EMMEANS) at P\u0026lt;0.05. Abbreviations: Mi= \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, Pp= \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, MP= \u003cem\u003eM. incognita\u003c/em\u003e + \u003cem\u003eP. penetrans\u003c/em\u003e, CETP=1-cyano-2,3-epithiopropane, CN= cyanide, ITC= isothiocyanate. *CYP83A1 also occurs in the benzenic glucosinolate biosynthesis pathway\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/ac1e0043fe5173046059d080.png"},{"id":86626654,"identity":"17ebe6c6-12d1-45e4-9dd7-b696c2f79550","added_by":"auto","created_at":"2025-07-14 05:30:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":254366,"visible":true,"origin":"","legend":"\u003cp\u003eContents of tryptophan, indole glucosinolates, and their breakdown products in \u003cem\u003eBrassica nigra\u003c/em\u003e roots, including the relative expressions of CYP79B2 gene and the transcription factor MYB122 as they occur in the indole glucosinolate metabolism pathway. Boxes and names in gray are parts of the pathway that were not measured in the experiment. Metabolites below or onto the right of the broken line are breakdown products. The biological replicates per treatment (n\u003csub\u003econtrol, Mi \u003c/sub\u003e= 5; n\u003csub\u003ePp, MP\u003c/sub\u003e = 3) are represented by points in the boxplots. Boxplots labeled with different letters are significantly different as per the estimated marginal means (EMMEANS) at P\u0026lt;0.05. Abbreviations: Mi= \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, Pp= \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, MP= \u003cem\u003eM. incognita\u003c/em\u003e + \u003cem\u003eP. penetrans\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/1b6745d9b1a3924b2a6740ce.png"},{"id":86626645,"identity":"e64b8294-9027-4f73-a40f-9c29818c4364","added_by":"auto","created_at":"2025-07-14 05:30:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":157909,"visible":true,"origin":"","legend":"\u003cp\u003eContents of phenylalanine, the glucosinolate gluconasturtiin, and the corresponding breakdown products in \u003cem\u003eBrassica nigra\u003c/em\u003e roots, including the relative expressions of CYP83A1 gene as they occur in the benzenic glucosinolate metabolism pathway. Boxes and names in gray are parts of the pathway that were not measured in the experiment. Compounds below the broken line are breakdown products. Boxplots labeled with different letters are significantly different as per the estimated marginal means (EMMEANS) at P\u0026lt;0.05. The biological replicates per treatment (n\u003csub\u003econtrol, Mi \u003c/sub\u003e= 5; n\u003csub\u003ePp, MP\u003c/sub\u003e = 3) are represented by points in the boxplots. Abbreviations: Mi= \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, Pp= \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, MP= \u003cem\u003eM. incognita\u003c/em\u003e + \u003cem\u003eP. penetrans\u003c/em\u003e, CN=cyanide, ITC= isothiocyanate. *CYP83A1 also occurs in the aliphatic glucosinolate biosynthesis pathway.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/f0d8aa9670ca2b73d53d6f46.png"},{"id":86626666,"identity":"526827f8-556c-4c1c-a173-ff52d15ea3e8","added_by":"auto","created_at":"2025-07-14 05:30:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":158519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e nematode performance assay showing nematode attraction (a-b) and early penetration in a Pluronic gel plate (c-e). The PPNs of interest were inoculated alone (Mi, Pp); inoculated two days after the other (Pp after Mi, Mi after Pp) or inoculated together (Mi in MP, Pp in MP). a and b show the proportion of nematodes Mi or Pp found touching the root at 4, 24, 48 and 72 hours after inoculation in Pluronic gel plates. The connected dark points denote the mean per treatment group per time point. Means that are marked with * are statistically different from the first counting time (4 hours) within each treatment group. Means connected by different letters denote significant difference of treatments per designated time point based on \u003cem\u003eWilcoxon’s pairwise test\u003c/em\u003e at P \u0026lt; 0.05 following \u003cem\u003eFriedman Rank Sum test\u003c/em\u003e.\u0026nbsp; The colored points represent the biological replicates per treatment group, \u003cem\u003en\u003c/em\u003e=10. The spread of ribbons denotes 95% confidence interval. c-e shows the number of nematodes that were found inside \u003cem\u003eB. nigra\u003c/em\u003e roots at three-day post inoculation via acid fuchsin staining. c-d presents a comparison of Mi (c) and Pp (d) penetration under different conditions: when inoculated concurrently, alone, or sequentially after the other species. For single-species plates (Mi alone, Mi after Pp, Pp alone, and Pp after Mi) 100 individuals were added to each plate. For dual-species plates (Mi in MP and Pp in MP) 50 individuals of each species were added (total of 100 nematodes per plate). The nematode counts were normalized to percentages by dividing the actual count of species X by the numbers of species X added to the plate (either 100 or 50). e illustrates the percentages of Mi and Pp that penetrated the roots of plants with concurrent PPN infection. The datasets were analyzed with the \u003cem\u003eGeneralized Linear Model\u003c/em\u003e (GLM). Boxplots labeled with different letters are significantly different as per the estimated marginal means (emmeans) at P\u0026lt;0.05. Boxplot legend: The box represents the interquartile range with the upper line representing the 75\u003csup\u003eth\u003c/sup\u003e percentile and the lower line representing the 25\u003csup\u003eth\u003c/sup\u003e percentile. The middle line represents the median. The whiskers extend to the (upper) maximum and (lower) minimum values, illustrating the overall spread of the data within one treatment. Black dots beyond the whiskers represent the outliers. Points represent the number of biological replicates per treatment, \u003cem\u003en\u003c/em\u003e=10. Abbreviations: Mi= \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, Pp= \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, MP= \u003cem\u003eM. incognita\u003c/em\u003e + \u003cem\u003eP. penetrans\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/66a8c813be03f0184016051f.png"},{"id":91358944,"identity":"08cca2b6-7731-41b3-b855-45593c330695","added_by":"auto","created_at":"2025-09-15 16:01:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1771469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/cb94654e-13e1-4ce0-bda6-ee976253fa6c.pdf"},{"id":86626644,"identity":"1c7443b1-6e57-4927-ab76-865b26fa319a","added_by":"auto","created_at":"2025-07-14 05:30:30","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3094239,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/b816a979e0aa25f687240441.xlsx"},{"id":86626661,"identity":"63f99fe6-4ee5-4221-a840-31e192522720","added_by":"auto","created_at":"2025-07-14 05:30:34","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":107556,"visible":true,"origin":"","legend":"","description":"","filename":"GelRNAextraction.png","url":"https://assets-eu.researchsquare.com/files/rs-6835102/v1/033974356044311bbc99f606.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual and single-species nematode infections distinctly modulate defense metabolism in Brassica nigra roots","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePlant-parasitic nematodes (PPNs) are root-feeding roundworms that pose significant threats to global agriculture. Several PPN species are causing extensive damage to crop plants (Nicol et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Jones et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tileubayeva et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with yield losses estimated at 173\u0026nbsp;billion USD worldwide (Kantor et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eMeloidogyne incognita\u003c/em\u003e and \u003cem\u003ePratylenchus penetrans\u003c/em\u003e are two prominent PPN species known for their detrimental effects on various crops (Jones et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kantor et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The root-knot nematode (RKN) \u003cem\u003eM. incognita\u003c/em\u003e is a sedentary endoparasitic nematode. The infective juvenile (J2) invades the roots by moving in-between cells until it reaches the vascular cylinder and induces the formation of giant cells. Thereafter, it becomes sedentary throughout its feeding and reproductive stages (Kyndt et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e; Bartlem et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rutter et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The giant cells serve as nutrient sinks, diverting resources away from other plant tissues and impairing its growth and productivity (Kyndt et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rutter et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mitchum et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, \u003cem\u003eM. incognita\u003c/em\u003e secretes effectors to modulate its host's immune responses and facilitate parasitism (Gheysen and Mitchum, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Eloh et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rutter et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, on the other hand, is a migratory endoparasitic nematode that feeds by puncturing individual cells and withdrawing nutrients as they move through the roots (Sijmons et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Fosu-Nyarko and Jones, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The effectors secreted by \u003cem\u003eP. penetrans\u003c/em\u003e enable mobility, causing tissue destruction due to, for example, cell wall degrading enzymes (Vieira et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Unlike RKNs, \u003cem\u003eP. penetrans\u003c/em\u003e does not induce the formation of giant cells, but it can cause extensive root damage leading to root necrosis, reduced nutrient and water uptake, as well as render the infected roots vulnerable to secondary microbial infections (Sijmons et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Fosu-Nyarko and Jones, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Several studies showed that plants can recognize and respond to nematode infections through various defense mechanisms. This includes activating defense-related genes and synthesizing secondary metabolites (Hofmann et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kyndt et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e; Desmedt et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies on related PPNs demonstrated that migratory and sedentary endoparasitic nematodes regulate plant defenses differently (Lohmann et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kyndt et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e). In rice, early root infection by the migratory endoparasitic nematode \u003cem\u003eHirsmaniella oryzae\u003c/em\u003e up-regulated biotic stress-responsive genes, inducing oxidative stress and programmed cell death. In contrast, early infection by the sedentary endoparasite, \u003cem\u003eMeloidogyne graminicola\u003c/em\u003e, suppressed root defense-responsive genes (Kyndt et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBrassica\u003c/em\u003e spp. plants are frequently challenged by PPNs (Li\u0026eacute;banas and Castillo, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Mukhopadhyay and Roy, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hol et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In a survey by Hol et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003eMeloidogyne\u003c/em\u003e spp. and \u003cem\u003ePratylenchus\u003c/em\u003e spp. were found to infest field-grown black mustard plants, \u003cem\u003eBrassica nigra\u003c/em\u003e. \u003cem\u003eBrassica\u003c/em\u003e plants are known to deploy glucosinolates as defense response against nematodes (van Dam et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Desmedt et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Glucosinolates (GSLs) are the main defense compounds of plants in the order Brassicales (Tsunoda et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Touw et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They are β-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-thioglucosides that can be classified by differences in their side chains and are grouped into aliphatic, benzenic, or indolic GSLs (Kliebenstein et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Agerbirk and Olsen, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Analyzing GSL profiles of nine GSL-producing plants including \u003cem\u003eB. nigra\u003c/em\u003e, Tsunoda et al. (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) showed that root GSL concentrations are significantly higher than in the shoots. Other than shoots, roots contain high levels of gluconasturtiin (2-phenylethyl GSL) (Tsunoda et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Touw et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sontowski et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which serves as defense against nematodes (Potter et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and insect herbivores, such as \u003cem\u003eDelia radicum\u003c/em\u003e and \u003cem\u003eD. floralis\u003c/em\u003e (Sontowski et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Several GSLs, either applied as plant extracts or incorporated as biofumigants, can reduce PPN populations and associated symptoms (Ren et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dahlin and Hallmann, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Eugui et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond the localized defense response, GSLs and other plant secondary metabolites are induced systemically in response to nematode infections, which correlated with altered performance of other herbivores feeding on the same plant (Hol et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; van Dam et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pajar et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Touw et al., 2025). Plant-mediated interactions between nematodes and aboveground insects, such as aphids and caterpillars (Hol et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; van Dam et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mbaluto et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and on root-feeding insects such as \u003cem\u003eD. radicum\u003c/em\u003e, have been reported (Touw et al., 2025). However, most studies have focused on responses to infections by single PPN species. This overlooks the fact that in natural environments, multiple PPN species interact within the same host plant (Hol et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mateille et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Sedentary and migratory PPN species may antagonize each other, whereby the population increase of one species suppresses the other (Gay and Bird, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Chapman and Turner, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Fontana et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It has been suggested that such effects are caused by competition for feeding sites or resources (Mateille et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Alternatively, antagonistic relationships between PPNs may be governed by plant-mediated mechanisms. For example, the phytohormones salicylic acid (SA) and jasmonic acid (JA) can be activated during PPN infections, which mainly depends on the PPN species interacting with the plant (Kyndt et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e; van Dam et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gheysen and Mitchum, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In general, SA is associated with systemic acquired resistance and is effective against biotrophic pathogens and sedentary PPNs (Bonnet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gheysen and Mitchum, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). On the other hand, JA is involved in induced responses that are commonly more effective against necrotrophic pathogens and migratory PPNs (Bonnet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Commonly, JA and SA act antagonistically via phytohormonal crosstalk (Pieterse et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Bonnet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gheysen and Mitchum, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). When a plant is simultaneously infected by several PPNs, JA-SA crosstalk may lead to a modified metabolic response, as it was found for chewing and phloem-feeding insects aboveground (Bonnet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This also prompts the question of whether the plant's defense response against concurrent (dual) nematode infections differs from those activated during single-species infections.\u003c/p\u003e \u003cp\u003eIn this study, we examined the changes in the root metabolome of \u003cem\u003eB. nigra\u003c/em\u003e when the plant is challenged by PPNs with contrasting feeding strategies: the migratory endoparasitic nematode \u003cem\u003eP. penetrans\u003c/em\u003e, and the sedentary endoparasitic nematode, \u003cem\u003eM. incognita.\u003c/em\u003e We hypothesized that the root metabolic changes in response to each nematode species are distinct due to their different feeding strategies. Moreover, we also analyzed root metabolic changes in response to the concurrent infection of \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans.\u003c/em\u003e We hypothesized that simultaneous infection would attenuate the defense response in comparison to single-species infections. Thus, the performance of one or both nematodes would improve on plants with simultaneous or prior infection by the other nematode. To test these hypotheses, we performed untargeted metabolic analysis on root samples infected with each nematode species as well as on root samples with concurrent infection. Our results indicate that root metabolic changes in response to concurrent infection by \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans\u003c/em\u003e are distinct from those observed in single-species infections, in particular for GSLs, lignans, and phenylpropanoids. Targeted analyses of GSLs and their breakdown products showed that sinigrin and allyl isothiocyanate (ITC) levels increased in \u003cem\u003eP. penetrans\u003c/em\u003e-treated plants, while gluconasturtiin and 2-phenylethyl ITC marginally increased in \u003cem\u003eM. incognita\u003c/em\u003e-infected plants. These GSLs and ITCs were reduced in MP-treated plants, along with indole GSLs, lignans and phenylpropanoids. Concurrent infection increased the number of \u003cem\u003eM. incognita\u003c/em\u003e inside the roots, while \u003cem\u003eP. penetrans\u003c/em\u003e numbers remained unaffected. However, fewer \u003cem\u003eP. penetrans\u003c/em\u003e were found in the roots when inoculated two days after \u003cem\u003eM. incognita\u003c/em\u003e. Our results highlight the importance of studying plant response to multiple nematode infections, as this may lead to changes in plant defense mechanisms that differ from those observed in single-species infections.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cem\u003ePlant Growing Conditions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBrassica nigra\u003c/em\u003e seeds were bulk-collected from a wild population at Elderveld, Arnhem, the Netherlands, in 2005. Prior to germination, the seeds were washed with 1% sodium hypochlorite solution and rinsed with ultrapure water. These clean seeds were germinated in water-soaked glass beads in plastic containers. The containers were covered with transparent plastic lids and kept in a climate chamber in a 16:8 (light: dark) photoperiod at 20:16\u0026deg; C (day: night). The seeds were germinated for 10 days before transplanting in sand pots (for the root material sampling) or Pluronic gel plates (for nematode-nematode interaction assay). The plant pots were prepared and maintained following the protocols described by van Dam et al. (2003). Before transplanting, each pot was filled with 2.5 l of dry, heat-treated sand (90 \u0026deg;C for 1 hour) and supplied with 200 ml tap water. The plants were grown in a greenhouse at 16:8-hour photoperiod, minimum light intensity 300 \u0026micro;mol m\u003csup\u003e-2 \u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; average temperature 25 \u0026deg;C; 60-80% relative humidity. The plants were supplied with 100 ml half-strength 3P Hoagland solution weekly. The developmental stages of \u003cem\u003eB. nigra\u003c/em\u003e were monitored following the universal BBCH scale (Lancashire et al., 1991).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eNematode Cultures\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePratylenchus penetrans\u003c/em\u003e was provided by the Plant Science Research Unit, Research Institute for Agriculture, Fisheries and Food (ILVO), Merelbeke, Belgium. The culture was maintained in carrot discs at 25 \u0026plusmn; 1 \u0026deg;C. \u003cem\u003eMeloidogyne incognita\u003c/em\u003e was provided by Bejo, Warmenhuizen, the Netherlands and was maintained on \u003cem\u003eSolanum lycopersicum\u003c/em\u003e cv. \u0026lsquo;Moneymaker\u0026rsquo; under greenhouse conditions (16:8 photoperiod at 25 \u0026plusmn; 3\u0026deg; C). All infective stages of \u003cem\u003eP. penetrans\u003c/em\u003e (juveniles and adults) and J2s of \u003cem\u003eM. incognita\u003c/em\u003e were extracted from the cultures via modified Baermann technique (Hooper et al., 2005). A solution containing a specified number of nematodes in water-Tween20\u0026reg; solution (0.04% v/v) was prepared and used for inoculation.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eGreenhouse Ex\u003c/em\u003eP\u003cem\u003eeriment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNematode inoculation\u003c/p\u003e\n\u003cp\u003eTo investigate nematode-induced changes in the roots, plants were assigned to the following treatment groups: control, \u003cem\u003eM. incognita\u003c/em\u003e only (Mi), \u003cem\u003eP. penetrans\u003c/em\u003e only (Pp), \u003cem\u003eM. incognita + P. penetrans\u003c/em\u003e inoculated simultaneously (MP). Four-week-old (BBCH 32) \u003cem\u003eB. nigra\u003c/em\u003e plants were inoculated with 2 ml of nematode suspension, each containing 200 nematodes in the infective stage in water-Tween20\u0026reg; solution. The same number of nematodes was applied in MP-treatments with 100 J2s of \u003cem\u003eM. incognita\u003c/em\u003e and 100 infective stages of \u003cem\u003eP. penetrans\u003c/em\u003e in 2 ml solution. Control plants were mock-inoculated with water-Tween20\u0026reg; solution. The nematode suspension was introduced into a small hole near the roots. Thereafter, 50 ml of tap water was added to facilitate nematode dispersal. The nematodes were allowed to infect for 10 days. A separate group of nematode-infected plants were left to grow for sixteen days more (total=26 days) to see the root galls and/or lesions, thereby confirming the success of nematode infection (Fig. S1).\u003c/p\u003e\n\u003cp\u003eRoot Sampling\u003c/p\u003e\n\u003cp\u003eOn the tenth day-post nematode inoculation (10 dpi), whole root samples were collected for metabolic (n\u003csub\u003e=\u003c/sub\u003e3-5) and gene expression (n=3-4) analyses. Plants were carefully lifted from their pots, and the roots were washed under running water. Excess water was drained, and the samples were collected, flash-frozen in liquid nitrogen, finely pulverized and stored at -80 \u0026deg;C freezer until further use. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eUntargeted Metabolomics via LC-MS coupled with MS/MS\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRoot metabolites were extracted using Liquid Chromatography-Time of Flight-Mass Spectrometry (LC-ToF-MS) according to Weinhold \u003cem\u003eet al. \u003c/em\u003e(2022). Twenty milligrams of ground freeze-dried root samples were mixed with 1 ml extraction solution (25% acetate buffer, pH 4.8 and 75% HPLC-grade MeOH). The mixture was placed in ultrasonic bath (5 minutes, 30Hz) then centrifuged at 15,000 x g for 15 minutes at room temperature. The supernatant was transferred to a new Eppendorf tube while the pellet was re-extracted with 1 ml of extraction solution, placed in ultrasonic bath (5 minutes, 30Hz) and centrifuged again at 15,000 x g for 15 minutes at room temperature. The supernatant was combined with that from the first extraction, then centrifuged at 15,000 x g for 10 minutes. Afterwards, 200 \u0026micro;l of supernatant was transferred in an HPLC vial and was added with 800 \u0026micro;l of extraction solution.\u003c/p\u003e\n\u003cp\u003eThe LC-MS analysis was conducted on an UltiMate\u0026trade; 3000 Standard Ultra-High-Performance Liquid Chromatography system (UHPLC, Thermo Scientific) with an Acclaim\u0026reg; Rapid Separation Liquid Chromatography (RSLC) 120 column (150 mm \u0026times; 2.1 mm, particle size 2.2 \u0026mu;m, ThermoFischer Scientific). The detailed instrument settings and post-processing parameters are provided in Method S1. After processing and blank feature-subtraction, the dataset contained 5,919 features. The resulting features with MS/MS data were annotated using an in-house spectral library. All detected features were classified and formatted using SIRIUS/CANOPUS (D\u0026uuml;hrkop et al., 2019; D\u0026uuml;hrkop et al., 2021; Hyun Woo Kim et al., 2021) and MetIgel v.1.0 \u0026copy;Smith and Schedl, 2021.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eTargeted Glucosinolate Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe glucosinolates (GSLs) were quantified via High-Performance Liquid Chromatography (HPLC, UltiMate\u0026trade; 3000, Thermo Scientific) using 50 mg freeze-dried ground root samples following the protocol by Grosser and van Dam (2017). The acquired data was further processed in Chromeleon 7.2 SR5 MUa (9624; Thermo Fisher Scientific, Waltham, MA, USA). \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eQuantification of GSL Breakdown Products\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGlucosinolate breakdown products were extracted following Hanschen and Schreiner (2017). Briefly, 25 mg of freeze-dried ground root sample was weighed into extraction vials and left to hydrolyze with 250 \u0026micro;l ultrapure water for 1 hour. Dichloromethane (DCM, 2 ml) were added along with 100 \u0026micro;l DCM containing 0.2 \u0026micro;mol benzonitrile (internal standard). The solution was shaken and centrifuged. The resulting DCM extract was dried over Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The extraction was repeated twice, adding only 1.5 ml DCM to the last two sets. The extracts were combined and reduced under nitrogen steam to 300 \u0026micro;l. The GSL breakdown products were quantified via Gas Chromatography-Mass Spectrometry (GC-MS, Agilent 7890 A Series GC System, Agilent Technologies) equipped with an Agilent 7683 Series Autosampler, an Agilent 7683B Series Injector and an Agilent 5975C inert XL MSD, using the settings as in (Hanschen, 2024).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eGene Expression Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative GSL biosynthesis and transport genes, and genes involved in phytohormone-mediated defenses were analyzed to assess the involvement of the respective processes in plant-nematode interactions. The genes and their short descriptions are as follows: \u003cem\u003eCYP83A1\u003c/em\u003e (CYTOCHROME P450, FAMILY 83, SUBFAMILY A, POLYPEPTIDE 1) is involved in the biosynthesis of aliphatic and benzenic GSLs. \u003cem\u003eCYP79B2\u003c/em\u003e (CYTOCHROME P450, FAMILY 79, SUBFAMILY B, POLYPEPTIDE 2) is involved in the conversion of tryptophan to indole-3-acetaldoxime, a precursor to IAA and indole GSLs; \u003cem\u003eMYB122\u003c/em\u003e (transcription factor MYB122) is known to regulate indole GSL biosynthesis; \u003cem\u003ePR1\u003c/em\u003e (pathogenesis-related protein 1) is a salicylic acid-responsive gene; \u003cem\u003ePAL1\u003c/em\u003e (phenylalanine ammonia-lyase 1) involved in the first reaction in the biosynthesis of secondary metabolites from L-phenylalanine; \u003cem\u003eERF1\u003c/em\u003e (ethylene response factor 1) encodes a transcription factor that can be activated by ethylene or jasmonates. The list of primer sequences used in this experiment is given in Table S1. Total RNA was extracted from 100 \u0026plusmn; 5 mg ground frozen root tissue following a protocol adapted from O\u0026ntilde;ate-S\u0026aacute;nchez and Vicente-Carbajosa (2008) as described in detail by Touw et al. (2020). The quality of DNAse I (Thermo Scientific, Waltham, MA, USA)-treated RNA was visually evaluated by gel-electrophoresis and by measurement of 260/230 nm and 260/280 nm absorbance ratios using a NanoPhotometer\u0026reg; P330 (Implen, Munich, Germany). Stable cDNA was synthesized from 4 \u0026mu;g purified total RNA using Revert Aid H minus reverse transcriptase (Thermo Scientific, Waltham, MA, USA) following the manufacturer\u0026rsquo;s instructions. The samples were incubated at 42 \u0026deg;C for 60 min, 50 \u0026deg;C for 15 min, and finally, 70 \u0026deg;C for 15 min in a thermal cycler (Techne, Stone, UK). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using the CFX384 Real-time system (BioRad, Munich, Germany) with gene-specific primers (Table S1). The qPCR conditions were: 2 min at 50 \u0026deg;C, 10 min at 95 \u0026deg;C, and 40 cycles of 15 sec at 95 \u0026deg;C and 1 min at 60 \u0026deg;C. Three technical replicates were analyzed per gene for each biological replicate (n= 3-4). The data was normalized to the average expression of the housekeeping genes \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eTIP41\u003c/em\u003e. The relative expression of target genes was calculated using the 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method as described in Livak and Schmittgen (2001).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eNematode-nematode Interaction Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRoot attraction assays were performed using a Pluronic gel medium, which simulates the three-dimensional soil environment (Wang et al., 2010). Following the protocol by Wang et al. (2009), the nematodes were mixed evenly into the Pluronic gel (Pluronic F-127, Sigma-Aldrich). One ml of gel was poured onto 3-cm Petri plates with 100 nematodes per plate. The plates were incubated at 27 \u0026deg;C for the gel to slightly solidify. The treatments were as follows: control (+ water), \u003cem\u003eM. incognita\u003c/em\u003e (Mi), \u003cem\u003eP. penetrans\u003c/em\u003e (Pp), and Mi + Pp inoculated simultaneously (MP) (n=10). To address the ambiguity of which PPN species infects the plant first, we included a sequential infection to see if prior infection of one species affects the other. For this, we added the treatments \u003cem\u003eM. incognita\u003c/em\u003e with \u003cem\u003eP. penetrans\u003c/em\u003e pre-infected plant (Mi after Pp, n=10) and \u003cem\u003eP. penetrans\u003c/em\u003e with \u003cem\u003eM. incognita\u003c/em\u003e pre-infected plant (Pp after Mi, n=10). \u003c/p\u003e\n\u003cp\u003eOne ten-day old \u003cem\u003eB. nigra\u003c/em\u003e seedling was placed in the middle of the plate. In plates with pre-infection (Mi after Pp; Pp after Mi), the first nematode species was allowed to infect for 48 h, whereafter the seedling was transferred to a plate containing the nematode of interest. Nematodes touching the roots were counted after 4, 24, 48 and 72 h. After counting at 72 h, the roots were stained with acid-fuchsin (Bybd et al., 1983) to count the nematodes that have entered the roots. For single-species plates (Mi alone, Mi after Pp, Pp alone, and Pp after Mi), we divided the number of nematodes by 100. For the data from dual-species plates (Mi in MP and Pp in MP), we divided the number of nematodes by 50. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans \u003c/em\u003ehave different feeding strategies and infection timelines. To facilitate uniformity in the context of concurrent infection, we defined early infection as the period from invasion until feeding site establishment for \u003cem\u003eM. incognita,\u003c/em\u003e and for \u003cem\u003ePratylenchus\u003c/em\u003e spp. the period before complete invasion of the vascular tissues, including early penetration and migration, which can occur three hours to three weeks post-inoculation (Acedo and Rohde, 1971; Hol et al., 2016; van Dam et al., 2018).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eStatistical Analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter data processing and compound annotation of the LC-MS data, the feature tables were exported to MetaboAnalyst 6.0 (Pang et al., 2024). The dataset was filtered based on their Interquartile Range. Given unequal replicate numbers and inherent variability among plants, Pareto-scaling was employed in addition to log\u003csub\u003e10\u003c/sub\u003e-transformation prior to the data analysis. The resulting dataset was analyzed using principal component analysis (PCA), followed by \u003cem\u003ePermutation Multivariate Analysis of Variance\u003c/em\u003e (PERMANOVA). Differentially abundant (DA) features based on pairwise comparisons between the control and each of the nematode-treated plants (Mi, Pp, MP) were identified using the Volcano Plot function in MetaboAnalyst 6.0. The lists were exported to InteractiVenn, creating Venn diagrams (Heberle et al., 2015). We grouped features at superclass level via NPClassifier (Hyun Woo Kim et al., 2021) in SIRIUS/CANOPUS (D\u0026uuml;hrkop \u003cem\u003eet al.\u003c/em\u003e, 2019, 2021) and assessed the difference in metabolite composition of nematode-treated roots using PERMANOVA via \u0026lsquo;adonis2\u0026rsquo; function in R-package vegan, with Euclidean distance. \u003c/p\u003e\n\u003cp\u003eThe nematode attraction assay was analyzed using \u003cem\u003eFriedman rank sum-test\u003c/em\u003e via the Friedman.test function in rstatix package (v4.1.2; R Core Team 2023) followed by post-hoc pairwise \u003cem\u003eWilcoxon test\u003c/em\u003e with \u003cem\u003eBonferroni\u003c/em\u003e \u003cem\u003ecorrection\u003c/em\u003e. Differences in nematode root penetration, as well as in GSLs, GSL breakdown products and gene expression data, were identified using generalized linear model (GLM) with the lme4 package in R (Bates et al., 2015). Data were Log-transformed to attain normal distribution when needed. In datasets that do not follow a Gaussian distribution based on visual (histogram) and statistical (\u003cem\u003eShapiro-Wilk Test\u003c/em\u003e) assessments, specific GLM distribution functions were used depending on the characteristics of each dataset (Bates et al., 2015). Estimated marginal means (emmeans) in R was used for post-hoc comparison. Figures and plots were generated in R with ggplot2 or in MetaboAnalyst 6.0 and were optimized for publication in Inkscape 1.1.1 (3bf5ae0d25, 2021-09-20, inkscape.org).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eEffects of Concurrent Nematode Infection on Root Metabolic Profiles\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrincipal component analysis\u003c/em\u003e (PCA) showed that the root metabolic profile of MP-infected plants differed distinctly from that of control, Mi- and Pp-infected plants (Fig. 1A, Table S2). This concurs with the observation that the roots of MP-treated plants had the most differentially accumulated (DA) features, both for up (352)- and down (210)-regulated features (562 in total; Fig. 1b, e-f). The roots of Mi-treated plants had the least up- (28) or down- (24) regulated features (Fig. 1c, e-f), followed by Pp-treatment (77 down- and 77 up-regulated features; Fig. 1d, e-f). A complete list of DA features is given in Table S3-a-c. Some DA features could be annotated using our in-house library. Among these annotated features, we found that Pp treatment uniquely up-regulated sinigrin levels (Fig. S2; Table S3-b). MP treatment uniquely down-regulated desulfo-4-hydroxyglucobrassicin and desulfo-glucobrassicin, and uniquely up-regulated cis-12-oxo-phytodienoic acid (OPDA) (Fig. S2; Table S3-c).\u003c/p\u003e\n\u003cp\u003eWe used the features that could be assigned to superclass level (858 of 5919 features) to analyze differences in the root metabolic composition of control and nematode-treated plants. A PERMANOVA showed that nematode treatment significantly affected root chemical composition (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 1.941, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.004; Fig. 2, Table S4). The nematode treatments explained approximately 32.7% of the variation in the dataset. The grouped features could be divided into three clusters (Fig. 2). The first cluster (labelled I in Fig. 2) included compound groups that are different in one or two treatment groups (Fig. 2). For example, tyrosine alkaloids (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 5.268, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.023) were reduced in Mi- and MP-treated plants, but unchanged in Pp-infected plants. \u0026nbsp;Nucleoside levels (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 3.608, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P\u0026nbsp;= 0.05) were slightly increased in Pp-infected plants and slightly reduced in MP-treated plants, whereas polycyclic aromatic polyketides (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 4.731, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.028) were reduced in Mi-treated plants. The second cluster is characterized by reduced peak intensity in MP-treated plants (Fig. 2-II). Among these are defense-related compound classes, in the shikimate pathway, such as lignans (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 6.05, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.007) and phenylpropanoids (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 5.517, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.01). Similarly, aromatic polyketides (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 9.271, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.002), peptide alkaloids (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 11.528, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.001), anthranilic acid alkaloids (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 3.303, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.05), pseudoalkaloids (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 5.886, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.01), and amino acid glycosides (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 4.895, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.031) were also significantly reduced in response to MP treatment. The NPClassifer-based classification assigned many GSLs to the amino acid glycoside class (Table S3-d). The third cluster is composed of compound groups with increased peak intensities in response to MP treatment. This includes sesquiterpenoids (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 5.446, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.021), linear polyketides (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 4.653, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.024), fatty acyl glycosides (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 8.650, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.003), and octadecanoids (\u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 66.028, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.004). Moreover, MP treatment also uniquely accumulated eight features classified as octadecanoids (Fig. 2, fourth row) and uniquely reduced four features classified as lignans (Fig. 1F; Fig. S2, second and third row; Table S3-b). A complete list of PERMANOVA results can be found in Table S4.\u003c/p\u003e\n\u003cp\u003eIn addition, we analyzed the expression levels of common marker genes related to defense signaling pathways, such as \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePAL1,\u003c/em\u003e and \u003cem\u003eERF1\u003c/em\u003e. The jasmonate/ethylene regulator \u003cem\u003eERF1\u003c/em\u003e was significantly down-regulated in all of the nematode-infected roots compared to the roots of control plants (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 23.114, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P \u0026lt; 0.001; Fig. S3). With marginal statistical significance, the SA-responsive gene \u003cem\u003ePR1\u003c/em\u003e was down-regulated in MP-treated roots compared to untreated roots (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 6.024, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.111, Fig. S3). \u003cem\u003ePAL1\u003c/em\u003e, however, was not differentially expressed by any of the nematode treatments (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 0.76, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.540). Unfortunately, our analyses of the JA-related gene yielded low expression values and therefore could not be reliably interpreted.\u003c/p\u003e\n\u003cp\u003eAltogether, these results suggest that metabolic changes in response to concurrent infection by \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans\u0026nbsp;\u003c/em\u003ediffered from the changes in response to each PPN species alone. The differences are reflected in many defense-related compound classes such as lignans, phenylpropanoids, octadecanoids, and GSLs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNematode-induced Changes in Root GSL and GSL Breakdown Products\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBased on the metabolic analyses, were we found the class amino acid glycosides, comprising many GSLs, significantly regulated, we performed targeted analyses of GSLs and their breakdown products. In addition, we analyzed the expression of GSL biosynthesis genes. The composition and relative abundance of GSL and breakdown products detected in the roots via targeted analysis is given in Table S5.\u003c/p\u003e\n\u003cp\u003eIn both targeted and untargeted analyses, we found that the levels of sinigrin were significantly higher in Pp-infected plants compared to control and MP-treated plants (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 10.511, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.015, Fig. 3). In line with this observation, we found a significant increase of the degradation product, allyl ITC, in roots of Pp-treated plants compared to other treatments (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 7.563, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.006; Fig. 3). The other breakdown products, 1-Cyano-2,3-epithiopropane (CETP) and allyl CN showed a similar pattern, though the differences were not statistically significant. Interestingly, the expression of \u003cem\u003eCYP83A1\u003c/em\u003e, involved in the synthesis of sinigrin, was significantly upregulated by Mi infection (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 3.67, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.05; Fig. 3) compared to Pp- and MP-infections.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the indole GSLs, particularly for glucobrassicin (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 5.098, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.017) and the neoglucobrassicin degradation product, 1-methoxyindole-3-acetonitrile (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 12.129, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.007), we found that they are significantly reduced in the roots of MP-treated plants compared to other treatment groups (Fig. 4). Showing a similar pattern, although not statistically significantly so, were the precursor amino acid, tryptophan (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 0.947, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.449), the GSLs downstream in the process, neoglucobrassicin (LM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 7.262, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.064), 4-hydroxyglucobrassicin (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 1.674, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.643), and 4-methoxyglucobrassicin (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 4.341, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.227), as well as the other indole GSL breakdown product, 4-methoxyindole-3-ACN (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 2.305, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.129). Moreover, \u003cem\u003eCYP79B2\u003c/em\u003e was significantly upregulated by Mi-treatment (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 3.74, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.049) compared to control and Pp-infected plants, while the transcription factor \u003cem\u003eMYB122\u003c/em\u003e was not differentially expressed by any of the nematode treatments (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 2.27, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.143) (Fig. 4).\u003c/p\u003e\n\u003cp\u003eSimilar to the indole GSLs, the levels of the benzenic GSL gluconasturtiin (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 22.128, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P \u0026lt; 0.001) and its degradation products, 2-phenylethyl ITC (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 3.477, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.05) and 2-phenylethyl CN (LM: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 4.753, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.021), had significantly lower levels in MP-treated plants (Fig. 5). Gluconasturtiin\u003cem\u003e\u0026nbsp;\u003c/em\u003elevels were significantly higher in Mi- (P \u0026lt; 0.001) and Pp-treated plants (P \u0026lt; 0.001) compared to MP-treated plants. In addition to its role in aliphatic GSL biosynthesis, the CYP83A1 gene is also involved in benzenic GSL biosynthesis. \u003cem\u003eCYP83A1\u003c/em\u003e is significantly downregulated in MP- and Pp-treated plants compared to Mi-treated plants (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 3.67, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;3, P = 0.05) (Fig. 5). The expression pattern of \u003cem\u003eCYP83A1\u003c/em\u003e was more similar to changes in this pathway than those in the aliphatic glucosinolate (sinigrin) pathways (Fig. 3).\u003c/p\u003e\n\u003cp\u003eThese results showed that single-species and concurrent nematode infections led to distinct changes in GSL and their breakdown products. In MP-treated plants, the levels of indole GSL and their breakdown products were reduced compared to Mi and PP plants. Both MP and Mi treatments altered the benzenic GSL, with marginally significant increased levels in response to Mi, yet significantly reduced levels in response to MP. Infection with Pp resulted in increased levels of sinigrin and its conversion products.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSimultaneous Infection Affected the Early Performance of Each Nematode Species\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe attraction of \u003cem\u003eM. incognita\u003c/em\u003e to uninfected roots differed from that of roots that were pre-infested or simultaneously infested with \u003cem\u003eP. penetrans\u0026nbsp;\u003c/em\u003e(Fig. 6a, Friedman \u0026chi;\u003csup\u003e2\u003c/sup\u003e = 75.685, \u003cem\u003edf\u003c/em\u003e = 4, P \u0026lt; 0.001; Fig. 6a). Over time, fewer \u003cem\u003eM. incognita\u003c/em\u003e touched the roots when the plants were pre-inoculated with Pp, in particular, compared to MP (M in MP, Wilcoxon test: 4 h P = 0.018, 24 h P = 0.018, 48 h P = 0.006, 72 h P = 0.017; Fig. 6a). Similarly, fewer \u003cem\u003eP. penetrans\u003c/em\u003e touched \u003cem\u003eB. nigra\u003c/em\u003e roots when plants were pre-infested with Mi (Fig. 6b, Friedman \u0026chi;\u003csup\u003e2\u003c/sup\u003e = 85.573, \u003cem\u003edf\u003c/em\u003e = 4, P \u0026lt; 0.001), especially when compared to plants where \u003cem\u003eP. penetrans\u0026nbsp;\u003c/em\u003ewas inoculated alone. By the end of the counting period, more \u003cem\u003eM. incognita\u003c/em\u003e were touching the roots of MP plants (Fig. 6a), while more \u003cem\u003eP. penetrans\u003c/em\u003e were touching the roots when inoculated alone (Fig. 6b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInoculating Mi after or with Pp affected the penetration success of Mi at 3 dpi (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 94.512, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;2, P \u0026lt; 0.001; Fig. 6c). We found more \u003cem\u003eM. incognita\u003c/em\u003e in roots of the MP-treated plants compared to Mi plants (EMMEANS: P \u0026lt; 0.001) or to roots pre-infected with \u003cem\u003eP. penetrans\u003c/em\u003e (Mi after Pp) (EMMEANS: P \u0026lt; 0.001; Fig. 6c). Single and concurrent inoculations also differentially affected the \u003cem\u003eP. penetrans\u0026nbsp;\u003c/em\u003epenetration (GLM\u003csub\u003eGamma\u003c/sub\u003e: \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 31.21, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;2, P \u0026lt; 0.001; Fig. 6d). The numbers of \u003cem\u003eP. penetrans\u003c/em\u003e found in the roots of dual-infected plants (P in MP) were significantly higher than in plants with prior \u003cem\u003eM. incognita\u003c/em\u003e infection (Pp after Mi, EMMEANS: P = 0.001; Fig. 6d). There were also significantly more \u003cem\u003eP. penetrans\u003c/em\u003e found in the roots of singly-infected plants (Pp alone) compared to Mi pre-infested plants\u003cem\u003e\u0026nbsp;\u003c/em\u003e(EMMEANS: P \u0026lt; 0.001; Fig. 6d). When both nematodes were inoculated at the same time (MP), we found significantly more \u003cem\u003eM. incognita\u0026nbsp;\u003c/em\u003ethan \u003cem\u003eP. penetrans\u003c/em\u003e in the roots (LM\u003csub\u003eGaussian\u003c/sub\u003e: \u003cem\u003eF\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e= 4.5134, \u003cem\u003edf\u003c/em\u003e =\u0026thinsp;1, P = 0.0477; Fig. 6e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, these results showed that concurrent infection distinctly affects the early infection success of each nematode species, whereby the effects depended on the species and sequence of infection.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTo understand how concurrent nematode infection affected the performance of each nematode species, we examined how infection by \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans\u003c/em\u003e, alone and together, affected root metabolic profiles, particularly GSL and their breakdown products. Overall, concurrent infection led to a distinctly different root metabolic profile compared to that of single-species infections. This was mainly due to differences in defense-related compounds, such as lignans, phenylpropanoids, and GSLs. The different GSL classes responded individually to the different nematode treatments; Mi treatment increased benzenic GSLs, Pp treatment increased aliphatic GSLs, and MP treatment reduced indole GSLs. Furthermore, we found that \u003cem\u003eM. incognita\u003c/em\u003e performs better when co-inoculated with \u003cem\u003eP. penetrans\u003c/em\u003e, whereas \u003cem\u003eP. penetrans\u003c/em\u003e performed worse when having to colonize a \u003cem\u003eM. incognita\u003c/em\u003e infected plant.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eConcurrent PPN Infection Affected Root Metabolome Differently than Single-species Infection\u003c/h2\u003e \u003cp\u003eIn natural environments, plants are often simultaneously attacked by multiple herbivores, resulting in complex interactions between signaling pathways (Bonnet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; van Dam et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mbaluto et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). When herbivores with contrasting feeding strategies attack, the response to one herbivore can antagonize the response to another due to hormonal crosstalk or defense compound interaction (Pieterse et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Bonnet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mbaluto et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, simultaneous attacks by aphids and caterpillars can weaken JA-mediated defenses, making plants more vulnerable to caterpillar damage (Soler et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ali and Agrawal, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our study examined the root metabolic changes in response to concurrent infection of two nematodes with contrasting feeding strategies \u0026ndash; the sedentary endoparasite, \u003cem\u003eMeloidogyne incognita\u003c/em\u003e and the migratory endoparasite, \u003cem\u003ePratylenchus penetrans.\u003c/em\u003e First, we hypothesized that each nematode species would elicit a specific root metabolic response. Our results showed that the overall root metabolic profiles of \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans\u003c/em\u003e infected plants overall did not significantly differ from each other, nor did they differ from that of uninfected control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). However, when looking into the differentially accumulated (DA) features in nematode-infected vs. control roots, we found several DA features responding to \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans\u003c/em\u003e, with more DA compounds uniquely responding to each species, than DAs shared among these treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f). \u003cem\u003ePratylenchus penetrans\u003c/em\u003e uniquely up-regulated sinigrin levels, which is a typical response to tissue damage in \u003cem\u003eBrassica\u003c/em\u003e spp. (Traw, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The continuous tissue damage caused by the migratory \u003cem\u003eP. penetrans\u003c/em\u003e may have caused the increased level of the toxic allyl ITC. This response is less expected of \u003cem\u003eM. incognita\u003c/em\u003e infections, as early \u003cem\u003eMeloidogyne\u003c/em\u003e spp. infection typically causes less tissue damage (Gheysen and Mitchum, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; van Dam et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, in the infection process, \u003cem\u003eMeloidogyne\u003c/em\u003e spp. secrete effectors to down-regulate plant defense responses (Gheysen and Mitchum, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kyndt et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe further hypothesized that concurrent infections of \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans\u003c/em\u003e attenuate changes in the root metabolic profile. Concurrent infection resulted in a distinct root metabolic profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Other than expected, double infected plants showed the highest numbers of significantly upregulated and downregulated DA features (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), indicating a more complex and intense metabolic response. Interestingly, the distinct changes in root metabolic profiles could be predominantly attributed to the differential accumulation of several defense compounds in response to MP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This may suggest that dual infection may have amplified the plant's defense responses, resulting in the simultaneous regulation of multiple defense-related pathways. The distinguished increase of OPDA levels and those of other octadecanoids during dual infection suggests that the octadecanoid pathway may have been involved in enhancing the production of defensive metabolites (Le\u0026oacute;n and S\u0026aacute;nchez-Serrano, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Papazian et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). On the other hand, dual infection also reduced the levels of lignans and phenylpropanoids, two distinct groups of compounds with critical roles in plant defense (Dixon et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ražn\u0026aacute; et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Both compound classes have been implicated in plant responses to the RKN \u003cem\u003eM. javanica\u003c/em\u003e in tomatoes, as demonstrated by the upregulation of transcripts encoding for enzymes involved in their biosynthesis (Kamali et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Compounds classified as alkaloids, \u003cem\u003ei.e.\u003c/em\u003e, peptide alkaloids, aromatic alkaloids, anthranilic acid alkaloids, as well as aromatic polyketides were also reduced in MP-treated roots. The reduction of these compounds suggests a more compromised plant defense under double infection. Moreover, amino acid glycosides, a class in which several GSLs are found, were also significantly reduced in response to MP treatment. GSLs are key defense compounds in \u003cem\u003eBrassica\u003c/em\u003e plants and are known for their defensive role against herbivores, including nematodes (Potter et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). This motivated thus to perform targeted analysis of GSLs and GSL breakdown products to further understand their role in the response to dual and single-species nematode infections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNematode Infection Affects Specific GSL Metabolic Pathways\u003c/h2\u003e \u003cp\u003eOur results demonstrated that single-species and concurrent infections by \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eP. penetrans\u003c/em\u003e led to distinct alterations in the root GSL profile of \u003cem\u003eB. nigra\u003c/em\u003e. The \u0026lsquo;mustard oil bomb\u0026rsquo; mechanism, where tissue damage triggers the GSL hydrolysis by the enzyme myrosinase (Abdel-Massih et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), produces toxic breakdown products that can affect insects, microbes, and nematodes (van Dam et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Eugui et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sontowski et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chekanai et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe levels of sinigrin, as well as its breakdown product, allyl ITC, were induced by \u003cem\u003eP. penetrans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests an induced response that may be associated with the direct and extensive mechanical damage inflicted by \u003cem\u003eP. penetrans\u003c/em\u003e on root tissues. Allyl ITC, in particular, was shown to reduce the motility and increase the mortality of \u003cem\u003eP. penetrans\u003c/em\u003e in a time- and dose-dependent manner (Chekanai et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In MP-infected plants, however, sinigrin and allyl ITC levels were significantly reduced compared to that of Pp-treated plants. This reduction suggests that the presence of \u003cem\u003eM. incognita\u003c/em\u003e may have interfered with the sinigrin-mediated response of \u003cem\u003eB. nigra\u003c/em\u003e towards \u003cem\u003eP. penetrans.\u003c/em\u003e Follow-up bioassays using mutants impaired in the production of sinigrin or allyl-ITC in their roots, could further elucidate the role of sinigrin and its breakdown products in the defense against \u003cem\u003eP. penetrans.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe benzenic GSL, gluconasturtiin and its degradation products 2-phenylethyl ITC and 2-phenylethyl CN were also reduced in MP-treated plants, whereas these compounds were slightly higher in Mi-treated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Indeed, gluconasturtiin and 2-phenylethyl ITC are among the GSLs deemed effective in suppressing nematode populations (Potter et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Eugui et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in particular those of \u003cem\u003ePratylenchus\u003c/em\u003e spp. (Potter et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Potter et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Our results thus align with previous reports that gluconasturtiin and 2-phenylethyl ITC play a role in mediating plant responses to nematode infections (Potter et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Eugui et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConcurrent infection reduced the less abundant indole GSLs and their breakdown products, particularly glucobrassicin and 1-methoxyindole-ACN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Root-knot nematode feeding leads to significant changes in plant root tissue, starting with the establishment of feeding sites that result in the formation of giant cells, creating root galls (Gheysen and Mitchum, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mbaluto et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Transcriptomic analysis of giant cells obtained via laser dissection has shown that the indole GSL biosynthesis gene, \u003cem\u003eCYP79B2\u003c/em\u003e was down-regulated in the giant cells but up-regulated in surrounding vascular tissues (Barcala et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This shows that the metabolic route involving indole GSL can be among the defense pathways responding to RKN infection and may be manipulated by the nematode for successful feeding. Our results point in a similar direction. We also found a significant upregulation of \u003cem\u003eCYP79B2\u003c/em\u003e in \u003cem\u003eM. incognita-\u003c/em\u003einfected roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Indole GSLs, in particular, are known for their anti-pathogen effects (Clay et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Increased indole GSL levels have also been correlated with response to aphid feeding (Kim and Jander, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pajar et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Aphids and nematodes, particularly, RKN, share similarities regarding their interactions with host plants. Both organisms use effectors to manipulate plant metabolic processes while creating their respective feeding sinks (Gheysen and Mitchum, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Z\u0026uuml;st and Agrawal, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Given this similarity, it is not surprising that indole GSL could be one of the metabolites that respond to \u003cem\u003eM. incognita\u003c/em\u003e infection. If indole GSLs indeed confer resistance to RKN, then the reduction of glucobrassicin and 1-methoxyindole-3-ACN in response to the MP treatment may have potentially benefited \u003cem\u003eM. incognita\u003c/em\u003e, which correlates to better early infection performance compared to single-species inoculation. However, further evaluation of the specific effects of indole GSL and the breakdown products is needed to assess their defensive role against nematodes.\u003c/p\u003e \u003cp\u003eOur results also showed that even the least abundant GSL breakdown products responded to nematode infection. In particular, we found a significant reduction of the less abundant 1-methoxyindole-3-ACN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and 2-phenylethyl CN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) in the roots with MP treatment compared to Mi-treated and control plants. Due to their high GSL concentrations, several \u003cem\u003eBrassica\u003c/em\u003e species are effective biofumigants (Dahlin and Hallmann, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Eugui et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Biofumigation incorporates chopped plant materials into the soil, releasing toxic GSL breakdown products with nematocidal effects. This process significantly reduced nematode populations (Potter et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Eugui et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), including over 60% reduction in galls and egg masses of \u003cem\u003eMeloidogyne\u003c/em\u003e spp. in greenhouse settings (Oliveira et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Curto et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile studies on the effect of GSL breakdown products mostly focused on the more abundant and biologically active ITCs, these results indicate that also nitriles/CNs may play a role in plant-PPN interactions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eInsights on the Relationship of M. incognita and P. penetrans in Concurrent Infection\u003c/h2\u003e \u003cp\u003eWe also considered the presence of nematodes in close proximity to the roots (touching the root, attraction) and inside the roots (penetration) as measures of early infection success. Our results suggest that \u003cem\u003eM. incognita\u003c/em\u003e is more successful when co-inoculated with \u003cem\u003eP. penetrans\u003c/em\u003e, while \u003cem\u003eP. penetrans\u003c/em\u003e is unaffected by co-inoculation but was more successful in the absence of prior \u003cem\u003eM. incognita\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This implies that concurrent PPN infection affects each nematode species differently. \u003cem\u003eMeloidogyne\u003c/em\u003e spp. and \u003cem\u003ePratylenchus\u003c/em\u003e spp. were reported to behave antagonistically in several plant pathosystems (Gay and Bird, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Chapman and Turner, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Fontana et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), but there are not many studies showing the possible mechanisms for this antagonism, particularly in relation to plant-mediated responses. We found that MP treatment, among other changes in root secondary metabolite, reduced indole (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and benzenic (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) GSLs, as well as their breakdown products. The reduction of specific GSL and their breakdown products, as well as the down-regulation of metabolites belonging to other defense-related classes, such as phenylpropanoids and lignans (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Table S4), suggest that the plant defenses are compromised under dual infection. The enhanced OPDA and other octadecanoids (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) levels in MP-treated plants suggest increased signaling in the JA pathway, which in turn may have suppressed the SA signaling pathway. Since \u003cem\u003eM. incognita\u003c/em\u003e is more susceptible to SA-based defenses (Bonnet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gheysen and Mitchum, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the metabolic changes in response to concurrent infection may have favored \u003cem\u003eM. incognita.\u003c/em\u003e This is associated with the greater early infection success of \u003cem\u003eM. incognita\u003c/em\u003e in concurrent infections compared to single-species infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This is, however, not the case for \u003cem\u003eP. penetrans\u003c/em\u003e, whose early infection success did not significantly differ when it was infected alone or with \u003cem\u003eM. incognita.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eWe observed an overall decline in the number of nematodes in contact with the roots over time, except in the Pp and Pp in MP treatment groups. The decline is likely due to the fact that \u003cem\u003eM. incognita\u003c/em\u003e J2 rapidly penetrate the roots and thus are not visible outside anymore. To confirm this, we stained the roots after counting at 72h. The stained roots of MP-treated plants showed that more \u003cem\u003eM. incognita\u003c/em\u003e were able to enter the roots at 3 dpi compared to \u003cem\u003eP. penetrans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). According to Mateille et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), PPN species may affect each other via competition when both species use the same limited resources. It is thus possible that, whereas the attenuated defense response favored \u003cem\u003eM. incognita\u003c/em\u003e in MP treatments, \u003cem\u003eM. incognita\u003c/em\u003e may have also outcompeted \u003cem\u003eP. penetrans\u003c/em\u003e in terms of limited resources, such as nutrients and infection sites. Though we did not include root metabolic measurements of the Pp-after-Mi treatment, the results of the nematode performance assay showed that \u003cem\u003eP. penetrans\u003c/em\u003e is negatively affected by prior \u003cem\u003eM. incognita\u003c/em\u003e infection. Nevertheless, more bioassays, particularly using mutants impaired in previously mentioned signaling pathways, are needed to elucidate the underlying mechanism of nematode-nematode interactions in concurrent infection.\u003c/p\u003e \u003cp\u003eTaken together, we showed that the root metabolic profile of dual nematode-infected \u003cem\u003eB. nigra\u003c/em\u003e is markedly different from those of plants infected by either \u003cem\u003eM. incognita\u003c/em\u003e or \u003cem\u003eP. penetrans\u003c/em\u003e alone. This suggests that the concurrent infection by both nematodes triggers a unique metabolic response that is not merely a combination of the individual responses to each nematode. This novel information generated from untargeted metabolic analysis could serve as basis for generating further hypotheses in exploring specific plant responses to single and dual nematode infections. In particular GSL and their breakdown products responded differently to the nematode treatments. Considering the specificity of the GSL response, we suggest to further explore the specificity of GSL and GSL breakdown products as defenses against different plant parasitic nematode species. This knowledge can potentially augment the effectiveness of biofumigation and other related crop protection strategies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eWe acknowledge support by the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig (funded by the German Research Foundation (DFG), grant no. FZT 118, 202548816), the International Max Planck Research School \u0026quot;Chemical Communication in Ecological Systems\u0026quot; and Leibniz Institute for Vegetable and Ornamental Crops (IGZ) for the funding to JAP and NMvD. We acknowledge the Erasmus\u0026thinsp;+\u0026thinsp;mobility fund - KA1 for the Erasmus Mundus Joint Master Degrees Programme of the European Commission under the PLANT HEALTH Project for providing support to PO, as well as the Erasmus\u0026thinsp;+\u0026thinsp;mobility fund and VLIR-OUS for the Masters in Agro- and Environmental Nematology program that supported AL.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJAP and NMvD conceptualized the study and designed the experiments. JAP and PO performed the greenhouse experiment, processed the root samples, and performed glucosinolate quantification. JAP and ALL optimized and performed the nematode performance assays. JAP and SD performed untargeted metabolomic analysis. JAP and FSH acquired and analyzed the data for glucosinolate breakdown products. JAP performed the gene expression analysis. JAP wrote the first draft of the paper with support from NMvD. All authors contributed to the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe would like to thank Dr. Rebekka Sontowski, Dr. Axel Touw, Maria Skoruppa, and Jessica Eichhorn for their assistance during the GSL and GSL breakdown product analyses. We also want to thank Prof. Nicole Viaene (Department of Plant Sciences, Flanders Institute for Agriculture, Fisheries and Food, Belgium) and Patricia Poot (Bejo BV, Warmenhuizen, The Netherlands) for providing us with nematodes.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData supporting this study are included in the manuscript and supplementary files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Massih RM, Debs E, Othman L, Attieh J, Cabrerizo FM (2023) Glucosinolates, a natural chemical arsenal: More to tell than the myrosinase story. Front Microbiol 14: 1\u0026ndash;15\u003c/li\u003e\n\u003cli\u003eAcedo JR, Rohde RA (1971) Histochemical Root Pathology of \u003cem\u003eBrassica oleracea capitata\u003c/em\u003e L. Infected by \u003cem\u003ePratylenchus penetrans\u003c/em\u003e (Cobb) Filipjev and Schuurmans Stekhoyen (Nematoda: Tylenchidae). J Nematol 3: 62\u0026ndash;68\u003c/li\u003e\n\u003cli\u003eAgerbirk N, Olsen CE (2012) Glucosinolate structures in evolution. Phytochemistry 77: 16\u0026ndash;45\u003c/li\u003e\n\u003cli\u003eAli JG, Agrawal AA (2014) Asymmetry of plant-mediated interactions between specialist aphids and caterpillars on two milkweeds. Funct Ecol 28: 1404\u0026ndash;1412\u003c/li\u003e\n\u003cli\u003eBarcala M, Garc\u0026iacute;a A, Cabrera J, Casson S, Lindsey K, Favery B, Garc\u0026iacute;a-Casado G, Solano R, Fenoll C, Escobar C (2010) Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells. Plant J 61: 698\u0026ndash;712\u003c/li\u003e\n\u003cli\u003eBartlem DG, Jones MGK, Hammes UZ (2014) Vascularization and nutrient delivery at root-knot nematode feeding sites in host roots. J Exp Bot 65: 1789\u0026ndash;1798\u003c/li\u003e\n\u003cli\u003eBates D, M\u0026auml;chler, MartinBates D, M\u0026auml;chler M, Bolker B, Walker S. 2015. Fitting Linear Mixed-Effects Models Using lme4. Journal of Statistical Software 67 1\u0026ndash;48., Bolker B, Walker S (2015) Fitting Linear Mixed-Effects Models Using lme4. J Stat Softw 67: 1\u0026ndash;48\u003c/li\u003e\n\u003cli\u003eBonnet C, Lassueur S, Ponzio C, Gols R, Dicke M, Reymond P (2017) Combined biotic stresses trigger similar transcriptomic responses but contrasting resistance against a chewing herbivore in \u003cem\u003eBrassica nigra\u003c/em\u003e. BMC Plant Biol 17: 1\u0026ndash;14\u003c/li\u003e\n\u003cli\u003eBybd DW, Kirkpatrick T, Barker KR (1983) An improved technique for clearing and staining plant tissues for detection of nematodes. J Nematol 15: 142\u0026ndash;143\u003c/li\u003e\n\u003cli\u003eChapman RA, Turner DR (1975) Effect of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e on Reproduction of \u003cem\u003ePratylenchus penetrans\u003c/em\u003e in Red Clover and Alfalfa. J Nematol 7: 6\u0026ndash;9\u003c/li\u003e\n\u003cli\u003eChekanai V, Neilson R, Roberts D, Edwards S, Back M (2024) In vitro nematicidal efficacy of brassica-derived isothiocyanates against the root lesion nematode, \u003cem\u003ePratylenchus penetrans\u003c/em\u003e. Nematology 26: 899\u0026ndash;908\u003c/li\u003e\n\u003cli\u003eClay NK, Adio AM, Denoux C, Jander G, Ausubel FM (2009) Glucosinolate Metabolites Required for an Arabidopsis Innate Immune Response. Science (80- ) 323: 95\u0026ndash;101\u003c/li\u003e\n\u003cli\u003eCurto G, Dallavalle E, Matteo R, Lazzeri L (2016) Biofumigant effect of new defatted seed meals against the southern root-knot nematode, \u003cem\u003eMeloidogyne incognita\u003c/em\u003e. Ann Appl Biol 169: 17\u0026ndash;26\u003c/li\u003e\n\u003cli\u003eDahlin P, Hallmann J (2020) New Insights on the Role of Allyl Isothiocyanate in Controlling the Root Knot Nematode \u003cem\u003eMeloidogyne ha\u003c/em\u003eP\u003cem\u003ela\u003c/em\u003e. Plants. doi: 10.3390/plants9050603\u003c/li\u003e\n\u003cli\u003evan Dam NM, Tytgat TOG, Kirkegaard JA (2009) Root and shoot glucosinolates: a comparison of their diversity, function and interactions in natural and managed ecosystems. Phytochem Rev 8: 171\u0026ndash;186\u003c/li\u003e\n\u003cli\u003evan Dam NM, Witjes L, Svato\u0026scaron; A (2003) Interactions between aboveground and belowground induction of glucosinolates in two wild Brassica species. New Phytol 161: 801\u0026ndash;810\u003c/li\u003e\n\u003cli\u003evan Dam NM, Wondafrash M, Mathur V, Tytgat TOG (2018) Differences in hormonal signaling triggered by two root-feeding nematode species result in contrasting effects on aphid population growth. Front Ecol Evol. doi: 10.3389/fevo.2018.00088\u003c/li\u003e\n\u003cli\u003eDesmedt W, Mangelinckx S, Kyndt T, Vanholme B (2020) A Phytochemical Perspective on Plant Defense Against Nematodes. Front Plant Sci. doi: 10.3389/fpls.2020.602079\u003c/li\u003e\n\u003cli\u003eDixon RA, Achnine L, Kota P, Liu CJ, Reddy MSS, Wang L (2002) The phenylpropanoid pathway and plant defence - A genomics perspective. Mol Plant Pathol 3: 371\u0026ndash;390\u003c/li\u003e\n\u003cli\u003eD\u0026uuml;hrkop K, Fleischauer M, Ludwig M, Aksenov AA, Melnik A V, Meusel M, Dorrestein PC, Rousu J, B\u0026ouml;cker S (2019) Brief CommuniCation SIRIUS 4: a rapid tool for turning tandem mass spectra into metabolite structure information. Nat Methods 16: 299\u0026ndash;302\u003c/li\u003e\n\u003cli\u003eD\u0026uuml;hrkop K, Nothias L-F, Fleischauer M, Reher R, Ludwig M, Hoffmann MA, Petras D, Gerwick WH, Rousu J, Dorrestein PC, et al (2021) Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra. Nat Biotechnol 39: 462\u0026ndash;471\u003c/li\u003e\n\u003cli\u003eEloh K, Sasanelli N, Maxia A, Caboni P (2016) Untargeted Metabolomics of Tomato Plants after Root-Knot Nematode Infestation. J Agric Food Chem 64: 5963\u0026ndash;5968\u003c/li\u003e\n\u003cli\u003eEugui D, Escobar C, Velasco P, Poveda J (2022) Glucosinolates as an effective tool in plant-parasitic nematodes control: Exploiting natural plant defenses. Appl Soil Ecol 176: 104497\u003c/li\u003e\n\u003cli\u003eFontana LF, Dias-Arieira CR, Mattei D, Severino JJ, Biela F, Arieira JDO (2015) Competition between \u003cem\u003ePratylenchus zeae\u003c/em\u003e and \u003cem\u003eMeloidogyne incognita\u003c/em\u003e on sugarcane. Nematropica 45: 1\u0026ndash;8\u003c/li\u003e\n\u003cli\u003eFosu-Nyarko J, Jones MGK (2016) Advances in Understanding the Molecular Mechanisms of Root Lesion Nematode Host Interactions. doi: 10.1146/annurev-phyto-080615-100257\u003c/li\u003e\n\u003cli\u003eGay CM, Bird GW (1973) Influence of Concomitant \u003cem\u003ePratylenchus brachyurus\u003c/em\u003e and \u003cem\u003eMeloidogyne\u003c/em\u003e spp. on Root Penetration and Population Dynamics. J Nematol 5: 212\u0026ndash;7\u003c/li\u003e\n\u003cli\u003eGheysen G, Mitchum MG (2011) How nematodes manipulate plant development pathways for infection. Curr Opin Plant Biol 14: 415\u0026ndash;421\u003c/li\u003e\n\u003cli\u003eGheysen G, Mitchum MG (2019) Phytoparasitic nematode control of plant hormone pathways. Plant Physiol 179: 1212\u0026ndash;1226\u003c/li\u003e\n\u003cli\u003eGrosser K, van Dam NM (2017) A straightforward method for glucosinolate extraction and analysis with high-pressure liquid chromatography (HPLC). JoVE (Journal Vis Exp 121: e55425\u003c/li\u003e\n\u003cli\u003eHanschen FS (2024) Acidification and tissue disruption affect glucosinolate and S-methyl-l-cysteine sulfoxide hydrolysis and formation of amines, isothiocyanates and other organosulfur compounds in red cabbage (\u003cem\u003eBrassica oleracea var. capitata f. rubra\u003c/em\u003e). Food Res Int 178: 114004\u003c/li\u003e\n\u003cli\u003eHanschen FS, Schreiner M (2017) Isothiocyanates, nitriles, and epithionitriles from glucosinolates are affected by genotype and developmental stage in \u003cem\u003eBrassica oleracea\u003c/em\u003e varieties. Front Plant Sci 8: 1\u0026ndash;17\u003c/li\u003e\n\u003cli\u003eHeberle H, Meirelles GV, Da Silva FR, Telles GP, Minghim R (2015) InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics 16: 169\u003c/li\u003e\n\u003cli\u003eHofmann J, El A, Ashry N El, Anwar S, Erban A, Kopka J, Grundler F (2010) Metabolic profiling reveals local and systemic responses of host plants to nematode parasitism. Plant J 62: 1058\u0026ndash;1071\u003c/li\u003e\n\u003cli\u003eHol WHG, Raaijmakers CE, Mons I, Meyer KM, van Dam NM (2016) Root-lesion nematodes suppress cabbage aphid population development by reducing aphid daily reproduction. Front Plant Sci 7: 1\u0026ndash;11\u003c/li\u003e\n\u003cli\u003eHooper DJ, Hallmann J, Subbotin SA (2005) Methods for extraction, processing and detection of plant and soil nematodes. Plant Parasit. nematodes Subtrop. Trop. Agric. Wallingford UK: CABI Publishing, pp 53\u0026ndash;86\u003c/li\u003e\n\u003cli\u003eHyun Woo Kim, Mingxun Wang, Christopher A. Leber, Louis-F\u0026eacute;lix Nothias, Raphael Reher, Kyo Bin Kang, Justin J. J. van der Hooft, Pieter C. Dorrestein, William H. Gerwick, Garrison W. Cottrell (2021) NPClassifier: A Deep Neural Network-Based Structural Classification Tool for Natural Products | Enhanced Reader. J Nat Prod 2795\u0026ndash;2807\u003c/li\u003e\n\u003cli\u003eJones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MGK, Kikuchi T, Manzanilla-L\u0026oacute;pez R, Palomares-Rius JE, Wesemael WML, et al (2013) Top 10 plant-parasitic nematodes in molecular plant pathology. Mol Plant Pathol 14: 946\u0026ndash;961\u003c/li\u003e\n\u003cli\u003eKamali S, Javadmanesh A, Stelinski LL, Kyndt T, Seifi A, Cheniany M, Zaki-Aghl M, Hosseini M, Heydarpour M, Asili J, et al (2022) Beneficial worm allies warn plants of parasite attack below-ground and reduce above-ground herbivore preference and performance. Mol Ecol 31: 691\u0026ndash;712\u003c/li\u003e\n\u003cli\u003eKantor M, Handoo Z, Kantor C, Carta L (2022) Top Ten Most Important U.S.-Regulated and Emerging Plant-Parasitic Nematodes. Hortic Rev. doi: 10.3390/horticulturae\u003c/li\u003e\n\u003cli\u003eKim JH, Jander G (2007) Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate. Plant J 49: 1008\u0026ndash;1019\u003c/li\u003e\n\u003cli\u003eKliebenstein DJ, Gershenzon J, Mitchell-Olds T (2001) Comparative Quantitative Trait Loci Mapping of Aliphatic, Indolic and Benzylic Glucosinolate Production in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e Leaves and Seeds. Genetics 159: 359\u0026ndash;370\u003c/li\u003e\n\u003cli\u003eKyndt T, Denil S, Haegeman A, Trooskens G, Bauters L, Van Criekinge W, De Meyer T, Gheysen G (2012a) Transcriptional reprogramming by root knot and migratory nematode infection in rice. New Phytol 196: 887\u0026ndash;900\u003c/li\u003e\n\u003cli\u003eKyndt T, Nahar K, Haegeman A, De Vleesschauwer D, H\u0026ouml;fte M, Gheysen G (2012b) Comparing systemic defence-related gene expression changes upon migratory and sedentary nematode attack in rice. Plant Biol 14: 73\u0026ndash;82\u003c/li\u003e\n\u003cli\u003eKyndt T, Vieira P, Gheysen G, de Almeida-Engler J (2013) Nematode feeding sites: Unique organs in plant roots. Planta 238: 807\u0026ndash;818\u003c/li\u003e\n\u003cli\u003eLancashire P, Bleiholde H, van Den Boom T, Langeluddeke P, Stauss R, Weber E, Witzenberger A. (1991) A uniform decimal code for growth stages of crops and weeds. Ann Appl Biol 119: 561-601.\u003c/li\u003e\n\u003cli\u003eLe\u0026oacute;n J, S\u0026aacute;nchez-Serrano JJ (1999) Molecular biology of jasmonic acid biosynthesis in plants. Plant Physiol Biochem 37: 373\u0026ndash;380\u003c/li\u003e\n\u003cli\u003eLi\u0026eacute;banas G, Castillo P (2004) Host suitability of some crucifers for root-knot nematodes in southern Spain. Nematology 6: 125\u0026ndash;128\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u0026minus;\u0026Delta;\u0026Delta;CT Method. Methods 25: 402\u0026ndash;408\u003c/li\u003e\n\u003cli\u003eLohmann M, Scheu S, M\u0026uuml;ller C (2009) Decomposers and root feeders interactively affect plant defence in Sinapis alba. Oecologia 160: 289\u0026ndash;298\u003c/li\u003e\n\u003cli\u003eMateille T, Tavoillot J, Goillon C, Pares L, Lef\u0026egrave;vre A, V\u0026eacute;die H, Navarrete M, Palloix A, Sage-Palloix A-M, Castagnone-Sereno P, et al (2020) Competitive interactions in plant-parasitic nematode communities affecting organic vegetable cropping systems. Crop Prot 135: 105206\u003c/li\u003e\n\u003cli\u003eMbaluto CM, Ahmad EM, Fu M, Mart\u0026iacute;nez-Medina A, van Dam NM (2020) The impact of \u003cem\u003eSpodo\u003c/em\u003ep\u003cem\u003etera exigua\u003c/em\u003e herbivory on \u003cem\u003eMeloidogyne incognita\u003c/em\u003e-induced root responses depends on the nematodes\u0026rsquo; life cycle stages. AoB Plants 12: plaa029\u003c/li\u003e\n\u003cli\u003eMbaluto CM, Vergara F, van Dam NM, Mart\u0026iacute;nez-Medina A (2021) Root infection by the nematode \u003cem\u003eMeloidogyne incognita\u003c/em\u003e modulates leaf antiherbivore defenses and plant resistance to \u003cem\u003eSpodoptera exigua\u003c/em\u003e. J Exp Bot 72: 7909\u0026ndash;7926\u003c/li\u003e\n\u003cli\u003eMitchum MG, Rocha RO, Huang G, Maier TR, Baum TJ, Hussey RS (2023) Genome-Guided Reanalysis of Root-Knot Nematode \u003cem\u003eMeloidogyne incognita\u003c/em\u003e Esophageal Gland Cell-Enriched Sequence Tag Libraries: A Resource for the Discovery of Novel Effectors. PhytoFrontiers. doi: 10.1094/PHYTOFR-09-22-0099-A\u003c/li\u003e\n\u003cli\u003eMukhopadhyay \u0026amp; Roy, K. AK (2006) Community analysis of major plant parasitic nematodes associated with vegetable crops in Eastern and Northeastern India. Int J Nematol 16: 194--199.\u003c/li\u003e\n\u003cli\u003eNicol JM, Turner SJ, Coyne DL, Nijs L Den, Hockland S (2011) Current nematode threats to world agriculture. Genomics Mol. Genet. Plant-Nematode Interact. pp 21\u0026ndash;43\u003c/li\u003e\n\u003cli\u003eOliveira RDL, Dhingra OD, Lima AO, Jham GN, Berhow MA, Holloway RK, Vaughn SF (2011) Glucosinolate content and nematicidal activity of Brazilian wild mustard tissues against \u003cem\u003eMeloidogyne incognita\u003c/em\u003e in tomato. Plant Soil 341: 155\u0026ndash;164\u003c/li\u003e\n\u003cli\u003eO\u0026ntilde;ate-S\u0026aacute;nchez L, Vicente-Carbajosa J (2008) DNA-free RNA isolation protocols for \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, including seeds and siliques. BMC Res Notes 1: 93\u003c/li\u003e\n\u003cli\u003ePajar JA, Otto P, Leonar AL, D\u0026ouml;ll S, van Dam NM (2024) Dual nematode infection in \u003cem\u003eBrassica nigra\u003c/em\u003e affects shoot metabolome and aphid survival in distinct contrast to single-species infection. Journal of Experimental Botany, Volume 75, 22:7317\u0026ndash;7336 \u003c/li\u003e\n\u003cli\u003ePang Z, Lu Y, Zhou G, Hui F, Xu L, Viau C, Spigelman AF, MacDonald PE, Wishart DS, Li S, et al (2024) MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res 52: W398\u0026ndash;W406\u003c/li\u003e\n\u003cli\u003ePapazian S, Girdwood T, Wessels BA, Poelman EH, Dicke M, Moritz T, Albrectsen BR (2019) Leaf metabolic signatures induced by real and simulated herbivory in black mustard (\u003cem\u003eBrassica nigra\u003c/em\u003e). Metabolomics 15: 1\u0026ndash;16\u003c/li\u003e\n\u003cli\u003ePieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5: 308\u0026ndash;316\u003c/li\u003e\n\u003cli\u003ePotter MJ, Davies K, Rathjen AJ (1998) Suppressive impact of glucosinolates in Brassica vegetative tissues on root lesion nematode Pratylenchus neglectus. J Chem Ecol 24: 67\u0026ndash;80\u003c/li\u003e\n\u003cli\u003ePotter MJ, Vanstone VA, Davies KA, Rathjen AJ (2000) Breeding to increase the concentration of 2-phenylethyl glucosinolate in the roots of \u003cem\u003eBrassica napus\u003c/em\u003e. J Chem Ecol 26: 1811\u0026ndash;1820\u003c/li\u003e\n\u003cli\u003eRažn\u0026aacute; K, N\u0026ocirc;žkov\u0026aacute; J, Vargaov\u0026aacute; A, Harenč\u0026aacute;r \u0026Aacute;, Bjelkov\u0026aacute; M (2021) Biological functions of lignans in plants. Agric 67: 155\u0026ndash;165\u003c/li\u003e\n\u003cli\u003eRen Z, Li Y, Fang W, Yan D, Huang B, Zhu J, Wang X, Wang X, Wang Q, Guo M, et al (2018) Evaluation of allyl isothiocyanate as a soil fumigant against soil-borne diseases in commercial tomato (\u003cem\u003eLycopersicon esculentum\u003c/em\u003e Mill.) production in China. Pest Manag Sci 74: 2146\u0026ndash;2155\u003c/li\u003e\n\u003cli\u003eRutter WB, Franco J, Gleason C (2022) Rooting Out the Mechanisms of Root-Knot Nematode-Plant Interactions. Annu Rev Phytopathol 60: 43\u0026ndash;76\u003c/li\u003e\n\u003cli\u003eSijmons PC, Atkinson H, Wyss U (1994) Parasitic Srategies of Root Nematodes and Associated Host Cell Responses. Annu Rev Phytopathol 32: 235\u0026ndash;59\u003c/li\u003e\n\u003cli\u003eSoler R, Badenes-P\u0026eacute;rez FR, Broekgaarden C, Zheng SJ, David A, Boland W, Dicke M (2012) Plant-mediated facilitation between a leaf-feeding and a phloem-feeding insect in a brassicaceous plant: From insect performance to gene transcription. Funct Ecol 26: 156\u0026ndash;166\u003c/li\u003e\n\u003cli\u003eSontowski R, Guyomar C, Poeschl Y, Weinhold A, van Dam NM, Vass\u0026atilde;o DG (2022) Mechanisms of Isothiocyanate Detoxification in Larvae of Two Belowground Herbivores, \u003cem\u003eDelia radicum\u003c/em\u003e and \u003cem\u003eD. floralis\u003c/em\u003e (Diptera: Anthomyiidae). Front Physiol 13: 1\u0026ndash;17\u003c/li\u003e\n\u003cli\u003eTileubayeva Z, Avdeenko A, Avdeenko S, Stroiteleva N, Kondrashev S (2021) Plant-parasitic nematodes affecting vegetable crops in greenhouses. Saudi J Biol Sci 28: 5428\u0026ndash;5433\u003c/li\u003e\n\u003cli\u003eTouw AJ, Mogena AV, Maedicke A, Sontowski R, van Dam NM, Tsunoda T (2020) Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in \u003cem\u003eBrassica rapa\u003c/em\u003e. Front Plant Sci 10: 1653\u003c/li\u003e\n\u003cli\u003eTraw MB (2002) Is induction response negatively correlated with constitutive resistance in black mustard? Evolution (N Y) 56: 2196\u0026ndash;2205\u003c/li\u003e\n\u003cli\u003eTsunoda T, Krosse S, van Dam NM (2017) Root and shoot glucosinolate allocation patterns follow optimal defence allocation theory. J Ecol 105: 1256\u0026ndash;1266\u003c/li\u003e\n\u003cli\u003eVieira P, Maier TR, Akker SEDEN, Howe DK, Zasada I, Baum TJ, Eisenback JD, Kamo K (2018) Identification of candidate effector genes of \u003cem\u003ePratylenchus penetrans\u003c/em\u003e. 9: 1887\u0026ndash;1907\u003c/li\u003e\n\u003cli\u003eWang C, Lower S, Thomas VP, Williamson VM (2010) Root-Knot Nematodes Exhibit Strain-Specific Clumping Behavior That Is Inherited as a Simple Genetic Trait. PLoS One 5: e15148\u003c/li\u003e\n\u003cli\u003eWang C, Lower S, Williamson VM (2009) Application of Pluronic gel to the study of root-knot nematode behaviour. Nematology 11: 453\u0026ndash;464\u003c/li\u003e\n\u003cli\u003eYu J, Vallad GE, Boyd NS (2019) Evaluation of allyl isothiocyanate as a soil fumigant for tomato (\u003cem\u003eLycopersicon esculentum\u003c/em\u003e Mill.) production. Plant Dis 103: 2764\u0026ndash;2770\u003c/li\u003e\n\u003cli\u003eZhang X, Song M, Gao L, Tian Y (2023) Metabolic variations in root tissues and rhizosphere soils of weak host plants potently lead to distinct host status and chemotaxis regulation of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e in intercropping. Mol Plant Pathol 1\u0026ndash;16\u003c/li\u003e\n\u003cli\u003eZ\u0026uuml;st T, Agrawal AA (2016) Mechanisms and evolution of plant resistance to aphids. Nat Plants 2: 15206\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"glucosinolates, nematode-nematode interactions, plant defense, root metabolome, simultaneous herbivory, systemic induced responses","lastPublishedDoi":"10.21203/rs.3.rs-6835102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6835102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant roots are exposed to various organisms that significantly impact plant productivity. Plant-parasitic nematodes (PPNs) such as \u003cem\u003eMeloidogyne\u003c/em\u003espp. and \u003cem\u003ePratylenchus \u003c/em\u003espp. are microscopic roundworms that damage several crops. In natural populations, \u003cem\u003eMeloidogyne\u003c/em\u003espp. and \u003cem\u003eP. penetrans\u003c/em\u003e were found to infest black mustard (\u003cem\u003eBrassica nigra\u003c/em\u003e) plants simultaneously. Considering their different feeding strategies and contrasting effects on plant defense responses, we hypothesized that dual infection may affect each nematode’s performance via changes in the root metabolome. Using untargeted and targeted metabolomics, we evaluated how single and dual nematode infections affected \u003cem\u003eB. nigra\u003c/em\u003e root metabolome. We combined these metabolic data with measures of early infection success. At three days post-inoculation, dual infection increased \u003cem\u003eM. incognita\u003c/em\u003e penetration success, while that of \u003cem\u003eP. penetrans \u003c/em\u003eremained unaffected. Compared to single-species infections, dual infections resulted in distinct root metabolic changes by reducing indole glucosinolates (GSL), gluconasturtiin, lignans, and phenylpropanoid levels. Dual and single-species infections affected different GSL classes. Sinigrin and its breakdown products increased in response to \u003cem\u003eP. penetrans, \u003c/em\u003ewhile \u003cem\u003eM. incognita \u003c/em\u003einfection increased gluconasturtiin and 2-phenylethyl ITC\u003cem\u003e. \u003c/em\u003eThis shows that plant defense response to dual nematode infection differ from those of single species, which has consequences to the early infection success of each nematode species.\u003c/p\u003e","manuscriptTitle":"Dual and single-species nematode infections distinctly modulate defense metabolism in Brassica nigra roots","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 05:30:19","doi":"10.21203/rs.3.rs-6835102/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-08T17:17:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T03:28:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-03T09:33:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55216282380446560395831682798813983284","date":"2025-06-16T20:31:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191829145399525655241471922562794065795","date":"2025-06-14T14:39:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-14T13:30:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-14T13:26:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-11T11:48:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Chemical Ecology","date":"2025-06-06T08:11:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aa556dd1-5857-4923-a081-0980a6db27ef","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T15:58:11+00:00","versionOfRecord":{"articleIdentity":"rs-6835102","link":"https://doi.org/10.1007/s10886-025-01637-8","journal":{"identity":"journal-of-chemical-ecology","isVorOnly":false,"title":"Journal of Chemical Ecology"},"publishedOn":"2025-09-10 15:56:55","publishedOnDateReadable":"September 10th, 2025"},"versionCreatedAt":"2025-07-14 05:30:19","video":"","vorDoi":"10.1007/s10886-025-01637-8","vorDoiUrl":"https://doi.org/10.1007/s10886-025-01637-8","workflowStages":[]},"version":"v1","identity":"rs-6835102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6835102","identity":"rs-6835102","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}