Meloidogyne incognita Significantly Alters the Cucumber Root Metabolome and Enriches Differential Accumulated Metabolites Regulating Nematode Chemotaxis and Infection

Abstract

Root-knot nematode (Meloidogyne incognita) is a globally destructive plant-parasitic nematode that severely impedes the sustainable production of horticultural crops. Metabolic reprogramming in plant roots represents the host response to M. incognita infection that can also be exploited by the nematode to facilitate its parasitism. In this study, untargeted metabolomics was employed to analyze metabolic changes in cucumber roots following nematode inoculation, with the goal of identifying differentially accumulated metabolites that may influence M. incognita behavior. Metabolomic analysis revealed that M. incognita significantly altered the cucumber root metabolome, triggering an accumulation of lipids and organic acids and enriching biotic stress-related pathways such as alkaloid biosynthesis and linoleic acid metabolism. Among differentially accumulated metabolites, myristic acid and hexadecanal were selected for further study due to their potential roles in nematode inhibition. In vitro assays demonstrated that both metabolites suppressed egg hatching and reduced infectivity of M. incognita, while pot experiments indicated a correlation between their application and reduced root gall formation. Chemotaxis assays further revealed that both metabolites exerted repellent effects on the chemotactic migration of M. incognita J2 and suppressed the transcriptional expression of two motility-and feeding-related neuropeptides, Mi-flp-1 and Mi-flp-18. In conclusion, this study demonstrates the significant potential of differentially accumulated metabolites induced by M. incognita infection for nematode disease control, achieved by interfering with nematode chemotaxis and subsequent infection. This work also provides deeper insights into the metabolomic mechanisms underlying the cucumber-M. incognita interaction.

1. Introduction

Root-knot nematodes, particularly Meloidogyne spp., are devastating soil pathogens of various crops and infect almost every species of vascular plants around the world [1]. Root-knot nematode (RKN) disease has been predominantly managed through the application of chemical nematicides and soil fumigants [2]. Traditionally, agronomic strategies such as rotational systems, intercropping, and cover crop cultivation represent established approaches for managing Meloidogyne spp. infestations [3,4,5]. Nematicides and soil fumigants are not only highly toxic to non-target soil organisms, such as earthworms, beneficial bacteria, and fungi, but also pose potential risks to human health [6]. These drawbacks underscore the urgent need for more sustainable, environmentally friendly, and targeted strategies to control RKN diseases.

As obligate sedentary nematodes, M. incognita form an intricate and sustained association with the root systems in host plants. This pathogenic interaction induces severe pathological damage through the establishment of specialized feeding sites (giant cells), which systemically impair vascular function, nutrient translocation, and water homeostasis essential for plant growth and development [7]. Chemotactic behaviors towards the roots of the host plant are critical for RKN infections. Generally, neuropeptide-mediated olfaction is a vital sensory modality that enables root-knot nematodes to sense host root exudate signals [8] and successful infection. The largest family of nematode neuropeptides is constituted by the FMRFamide-like peptides (FLPs), which are involved in multiple parasitic activities [9]. Our previous study demonstrated that the Mi-flp-1 and Mi-flp-18 genes (encoding FMRFamide-like peptides) regulated M. incognita chemotaxis towards cucumber and controlled infection using an in vitro RNAi assay [10]. There were also findings shown in several previous studies confirming the importance of Mi-flp-1 and Mi-flp-18 in regulating the chemotaxis and infection of M. incognita [10] and Meloidogyne graminicola [11].

During RKN parasitism, the infective second-stage juveniles (J2s) initiate parasitism by penetrating the root tip zone, where the protective tissues are often less developed. Following entry, these motile J2s navigate intercellularly through the root cortex, actively seeking suitable cells within the vascular cylinder to establish specialized, permanent feeding sites known as giant cells [12]. Following invasion of the host root system, M. incognita induces the formation of feeding sites within the vascular tissues to serve as a nutrient source for its development [13].

During this process, root-knot nematodes not only alter the production and allocation of metabolites in root tissues but also activate defense mechanisms in the host root system [12]. Previous study reviewed the chemical class against root-knot nematodes include terpenoids, flavonoids, and glucosinolates [14]. And phenolic acids were usually considered as potential nematocidal metabolites in plant parasitic nematodes management [15]. Studies indicate that root-knot nematode infection triggers a rearrangement of amino acid and sugar metabolic pathways in tomato root tissues [16]. Furthermore, complex alterations occur in both primary and secondary metabolic pathways of the host root system after nematode invasion, with the production of secondary metabolites potentially being a key factor in conferring nematode resistance [17,18]. Plants possess broad defense mechanisms against biotic stress, and the remodeling of root metabolism represents a crucial aspect of the plant’s response and defense against root-knot nematode stress. Metabolomics, the comprehensive analysis of global metabolite changes in plant samples [19], serves as a vital tool for studying plant responses to biotic stress [20]. Upon pathogen infection, numerous immune defense responses are directly mediated by secondary metabolites. Moreover, systemically induced plant-wide defenses can influence biological and metabolic alterations, thereby enhancing plant resilience to biotic stress [21]. Therefore, in-depth investigation into the metabolic changes in cucumber (Cucumis sativus) during nematode infection is essential for identifying potential resistance pathways and key secondary metabolites.

Cucumber (Cucumis sativus) stands as one of the most economically significant fruit vegetables cultivated under protected facilities globally [22]. However, challenges in soil renewal and prevalent continuous monoculture practices lead to the accumulation of soil-borne pathogens, severely constraining crop yield and quality. Among these, the southern root-knot nematode M. incognita is a particularly devastating plant-parasitic nematode, posing a major threat to the sustainable production of horticultural crops worldwide.

Evidence suggests that metabolic reprogramming within host roots is a key feature of the plant response and defense mechanisms against M. incognita infection [23]. Metabolic study of rice infected by nematodes enhanced nutrient transport and upregulated the biotic stress-related genes early in the infection [24]. To elucidate these metabolic interactions in cucumber, this study inoculated M. incognita into native soil. Roots sampled 14 days post-inoculation were subjected to untargeted metabolomic analysis to identify infection-induced metabolic changes and screen for differential metabolites with potential roles in regulating nematode activity. Building on the metabolomic findings, the impact of identified differential metabolites on M. incognita activity (including egg hatching and juvenile motility) and root infectivity was rigorously assessed. Furthermore, specific root-derived metabolites, identified as significantly altered, were evaluated at varying concentration gradients for their direct effects on nematode activity and their efficacy in suppressing cucumber root infection. This research aims to provide a scientific foundation for developing novel strategies to control M. incognita and inform the breeding of nematode-resistant cucumber cultivars.

2. Materials and Methods

2.1. Sampling Sites and Plant Materials

The root-knot nematode (M. incognita) used in this study was maintained on cucumber (Cucumis sativus L.) plants cultivated in autoclaved soil within greenhouse pots. Egg masses produced by the nematodes were carefully collected and transferred to Petri dishes for hatching to obtain second-stage juveniles (J2s) for subsequent inoculation. The cucumber cultivar used was ‘Zhongnong No. 26’. Seeds were surface-sterilized, germinated, and then sown in square plastic pots (10 cm × 8 cm) filled with soil to a depth of 10 cm. The experimental soil was collected from a cultivation field with no history of root-knot nematode disease. Prior to use, the soil was sieved through a 3 mm mesh to remove debris and ensure homogeneity. Key physicochemical properties of the prepared soil were as follows: total nitrogen, 3.12 g/kg; available phosphorus (Olsen-P), 70.20 mg/kg; available potassium (exchangeable K), 230.65 mg/kg; organic matter content, 30.17 g/kg; pH (1:2.5 soil–water), 7.25; and bulk density, 1.22 g/cm³.

2.2. Experimental Design

The experiment was conducted in the research greenhouse of the School of Life Sciences, Linyi University, Linyi City, Shandong Province, China. Prepared experimental soil with no history of root-knot nematode disease was filled into planting pots (Figure S1). Cucumber seeds were surface-sterilized and germinated on sterile filter paper within Petri dishes. Upon radicle emergence, seeds were dibbled into the planting pots. When cucumber seedlings reached the two-true-leaves stage, each plant was inoculated with approximately 300 s-stage juveniles (J2s) of the M. incognita. The inoculum was applied near the root system at a depth of approximately 1 cm. Control plants were mock-inoculated with an equivalent volume of sterile water. All treatments received identical cultivation management practices. Root samples were collected 14 days after-inoculation (DAI). This time point was selected as it represents the stage where nematodes have successfully infected roots and established feeding sites but have not yet completed their parasitic life cycle within the roots. At sampling, root tissues from every two cucumber plants were pooled to form one biological replicate. Six biological replicates were established per treatment. These pooled root samples were then subjected to untargeted metabolomic profiling.

2.3. Untargeted Metabolomic Profiling of Cucumber Roots Under M. incognita Stress

Cucumber plants at 14 DAI with M. incognita were carefully removed from their pots. Root systems were swiftly rinsed with sterile water to remove adhering soil particles and then immediately flash-frozen in liquid nitrogen. The total root system was harvested from the pots after removing large soil aggregates by shaking the roots. And then cucumber roots were put into a 50 mL tube filled with the cold solution that contained 50% methanol (v/v) with 0.05% formic acid (v/v) to remove the metabolites in adhering soil particles [25]. After shaking at 4 °C for 10 min, the root tissues were separated, put into liquid nitrogen immediately, and saved at −80 °C for further analysis. The frozen root samples were subsequently conducted by OE Biotech Company (Shanghai, China). The root tissues were homogenized in liquid nitrogen and an 80 mg accurately weighed sample was transferred to a 1.5 mL Eppendorf tube. After adding 1 mL methanol/water (4/1, v/v) and two 3 mm steel balls, the root samples were ground at 60 Hz for 2 min. The supernatant was collected and transferred to LC vials. Six biological replicates were examined, and each replicate had three roots. Untargeted metabolomic analysis in both ESI positive and ESI negative ion modes was performed using an ACQUITY UHPLC system (Waters Corporation, Milford, CT, USA) coupled with an AB SCIEX Triple TOF 5600 System (AB SCIEX, Framingham, MA, USA). Briefly, 5 μL of each sample was injected onto an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) and the binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v). The flow rate was 0.4 mL/min and column temperature were 45 °C. Data acquisition was performed in full scan mode (m/z ranges from 70 to 1000) combined with IDA mode. The acquired LC-MS raw data were identified by the progqenesis QI software 2.1 (Waters Corporation, Milford, CT, USA). Compound identification was performed by matching acquired mass spectra and retention times against reference spectra in the following metabolite databases: The Human Metabolome Database (HMDB, http://www.hmdb.ca/ accessed on 1 March 2025), Lipid Maps (v2.3, http://www.lipidmaps.org/ accessed on 1 March 2025), and METLIN (http://metlin.scripps.edu accessed on 1 March 2025). Metabolites exhibiting statistically significant changes (p-value ≤ 0.05) were defined as differential metabolites. These differential metabolites were then functionally annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to map them onto biological pathways.

2.4. Effects of Candidate Metabolites on Eegg Hatch and Mortality of the M. incognita

Specific differential root-derived metabolites, obtained commercially, were dissolved to create a concentration gradient series: 0, 0.2, 0.4, 0.6, 0.8, 1, 2, and 4 mM. For the egg hatch assay: Egg masses of the M. incognita were carefully selected. Eggs were separated into a suspension of individual eggs using a 0.05% sodium hypochlorite solution. Approximately 100 eggs were transferred into a 1.5 mL microcentrifuge tube. After centrifugation and removal of the supernatant, 1 mL of the metabolite solution at the respective test concentration was added to the pelleted eggs. The tubes were incubated at 28 °C. The egg hatch rate was determined 48 h post-treatment. Each concentration was replicated six times. For the juvenile mortality assay: Approximately 100 s-stage juveniles (J2s) were transferred into 1.5 mL microcentrifuge tubes. Following centrifugation and removal of the supernatant, 1 mL of the metabolite solution at the test concentration was added to the pelleted J2s. The tubes were incubated at 28 °C. After 48 h of exposure, nematode viability was assessed under an optical stereomicroscope. Actively moving J2s were counted as alive, while immobile J2s failing to respond to mechanical probing were considered dead. The nematode mortality rate was calculated to assess the inhibitory effect of the different metabolite concentrations on J2 activity.

2.5. Evaluation of Candidate Metabolites to M. incognita Infection on Cucumber

To evaluate the effect of candidate root-derived metabolites on nematode infection of cucumber roots. We conducted pot experiments following the methods described in Section 2.2. To minimize the influence of soil and better demonstrate the effects of metabolites, cucumber seeds were sterilized and sowed in 5 × 5 cm pots full of sterilized substrates (sand–vermiculite, 1:1, v/v) [10]. Cucumber seedlings having two true leaves were treated with different gradient of metabolites and inoculated with 100 M. incognita pre-J2s per seedling. The root galls were analyzed by the cucumber fresh root weight. For concentration gradient treatment, there were six biological replicates with independent plants.

2.6. Nematode Attraction Assay Under the Effects of Differentially Accumulated Metabolites

To examine whether the differentially accumulated metabolites have the potential to regulate the chemotaxis of M. incognita, we used a one-choice attraction assay that was adapted from the previous study [10]. Specifically, a 7 cm diameter Petri dish was divided into three distinct sections: the upper root tip zone (RTZ) containing a fresh cucumber root tips (1 cm) without metabolite treatment served as the negative control (denoted as ‘0’ in figures) or metabolite concentration gradients; the central 1 cm inoculation zone (IZ) was set for M. incognita inoculation; and the movement zone (MZ) was located in the lower area of Petri dish which represented the potential migration area for nematodes under repellent effects. The Petri dish was full of 23% (wt/vol, mixed overnight at 4 °C) Pluronic F-127 gel (Sigma-Aldrich) for nematode movement. When conducting experiments, 200 μL F-127 gel containing 100 M. incognita J2s was placed into the IZ. At 24 h after inoculation, the M. incognita J2s were observed using a stereomicroscope and counted to compare the ratio of M. incognita J2s in three different zones. The positive control and each treatment concentration gradient were established with six biological replicates.

2.7. Analysis of Mi-flp-1 and Mi-flp-18 Gene Expression in Nematode Attraction Assay

To investigate the effects of candidate metabolites on the expression of Mi-flp genes, total RNA was extracted from M. incognita J2s, which were separated from the one-choice attraction assay, to analyze the relative expression pattern of Mi-flp genes, including Mi-flp-1 (AY729023) and Mi-flp-18 (AY729022) [10,26]. Total RNA was extracted from the nematodes using the Eastep Super Total RNA Extraction Kit according to the manufacturer’s protocol (Promega, Madison, WI, USA). First-strand cDNA was synthesized from 500 ng RNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). qPCR was carried out with SYBR Green PCR Master Mix (Vazyme, Nanjing, China) in an ABI 6500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative target gene expression was analyzed using the comparative Ct method. M. incognita actin (accession BE225475) was taken as a reference gene for the normalization of gene expression. The primers for qPCR are listed in Table S2. Six technical replicates were performed for each of the samples during qPCR analysis.

2.8. Statistical Analysis

Multivariate statistical analysis of the untargeted metabolomics data was conducted using the R software (4.4.3, https://www.r-project.org/, accessed on 28 February 2025) environment. One-way analysis of variance (ANOVA) was performed using SPSS Statistics software (version 26.0). The results are expressed as mean ± SEM, where ANOVA results indicated significant differences (p < 0.05), and post hoc comparisons among group means were carried out using Tukey’s HSD test, following confirmation of homogeneity of variance. Data visualization was performed using GraphPad Prism 8.0.2 software (GraphPad Software, Boston, MA, USA).

3. Results

3.1. Effect of M. incognita Infection on Root Metabolome of Cucumber

Fourteen days after the roots of cucumber plants were infected with M. incognita, an untargeted metabolomic analysis of the cucumber roots was performed using liquid chromatography–mass spectrometry (LC-MS). Principal component analysis (PCA) of the metabolic profiles obtained from the samples effectively characterized the impact of nematode infection on the root metabolome. As shown in Figure 1, the base peak chromatograms of infected roots differed markedly from those of the control group. The first principal component (PC1) and second principal component (PC2) explained 24% and 18.4% of the total variance, respectively, and clear separation was observed between the two treatment groups. These results indicate that M. incognita infection induces significant changes in the overall metabolite profile of cucumber roots.

Partial Least Squares Discriminant Analysis (PLS-DA) is a supervised multivariate statistical analysis method. Figure 2 demonstrates that M. incognita infection significantly altered the metabolic profiles of cucumber roots between the two treatment groups. The R² value of 0.839 indicates a robust model fit. And the Q² value of 0.244 indicates that our PLS-DA model demonstrates modest but statistically meaningful predictive capability.

Furthermore, the volcano plot analysis, based on t-tests, revealed 22 significantly upregulated differential metabolites. These metabolites are likely induced by M. incognita infection and may possess the potential to influence the infectivity vigor of the root-knot nematode.

As shown in the bubble plot, the x-axis represents the RichFactor, which indicates the ratio of the number of differential metabolites enriched in a certain pathway to the total number of metabolites annotated in that pathway in the background database. M. incognita infection may alter metabolic pathways in cucumber roots. Concurrently, both primary and secondary metabolites within the cucumber root system are likely involved in the response to nematode stress. As revealed in Figure 3, multiple metabolic pathways were enriched during M. incognita infection. The significant enrichment of alkaloid biosynthesis pathways suggests that the plant may be enhancing the production of defensive alkaloids. The changes in lipid metabolism pathways indicate adjustments in membrane-related processes and signaling. Amino acid and other primary metabolic pathways are also involved, which may support the plant’s defense-related physiological and biochemical changes.

3.2. Screening of Root-Derived Differential Metabolites in Cucumber Root

The heatmap visually illustrates the alterations in various differential metabolites following the M. incognita infection of cucumber roots (Figure 4; Table S1). Categorization of these metabolites revealed that lipids and lipid-like molecules constituted the most prominent class of differentially expressed metabolites in cucumber root tissues under nematode stress. Within the root system, organic acids and derivatives and organoheterocyclic compounds also exhibited substantial changes.

From the upregulated differential metabolites in cucumber roots induced by M. incognita, two metabolites—myristic acid and hexadecanal—were selected based on their Variable Importance in Projection (VIP) scores derived from PLS-DA analysis. Corresponding standard compounds of these metabolites were purchased. Subsequently, we assessed the effects of different concentration gradients of these compounds on egg hatching, nematode mortality, and the infection capability of M. incognita on cucumber roots. Two compounds were selected for further analysis on the basis of the criteria of being synthesizable and inexpensive.

3.3. Effects of Candidite Metabolites on Egg Hatching and J2 Mortality of M. incognita

The effects of different metabolites and their concentration gradients on the egg hatching and second-stage juvenile (J2) mortality of M. incognita varied. Figure 5a shows that the egg mass hatch rate decreased with increasing concentrations of the two metabolites. Myristic acid induced a significant decline in hatch rate at 0.4 mM, while hexadecanal caused significant declines at 0.6 mM compared with CK. At the highest tested concentration of 4 mM, the nematode egg hatch rate reached its minimum, indicating the most pronounced inhibitory effect of all two metabolites on M. incognita egg hatching at this concentration.

Further, we have also examined the effects of different metabolites on the mortality of second-stage juveniles, and this holds practical significance for future field applications. Similarly, Figure 5b demonstrates that J2 mortality increased with metabolite concentration. A significant increase in mortality was observed for myristic acid at 0.2 mM, and for hexadecanal at 0.4 mM. Mortality peaked at 4 mM, signifying the greatest nematicidal efficacy of the metabolites against J2 larvae at this concentration.

3.4. Effects of Different Metabolites on M. incognita Infection in Cucumber

Further, regarding the invasion ability of M. incognita J2s, we used pot experiments to simulate the invasion of M. incognita into cucumber roots in actual field cultivation. The results showed that after the addition of the two metabolites, with the concentration gradient, the number of root galls on cucumber roots decreased significantly. In particular, the medium–high concentrations of myristic acid could significantly reduce the number of root galls by up to 50% after being applied to the cucumber root zone. After hexadecanal was applied to cucumber roots, its effect on inhibiting root-knot nematode infection was slightly weaker than that of myristic acid. However, at high concentrations such as 1, 2, and 4 mM, it could still significantly reduce the number of root galls (Figure 6).

Chemotaxis is a critical step for successful infection of M. incognita. We used a one-choice attraction assay to evaluate the effects of candidate metabolites on M. incognita J2s movement and associated gene expression. As shown in Figure 7a, the greater the accumulation in the RTZ, the more pronounced the chemotactic migration of M. incognita second-stage juveniles (J2). Results demonstrate that establishing concentration gradients of different candidate metabolites around cucumber root tips significantly disrupted the chemotactic migration capability of the root-knot nematode J2 towards the roots. In the RTZ, which contains root tips and metabolites, progressively fewer M. incognita second-stage juveniles (J2) migrated towards this area with increasing concentrations of myristic acid in the established gradient. In control treatments (cucumber root tips only), nematodes within the RTZ accounted for 65% of the total, whereas the addition of myristic acid, particularly at a high concentration (4 mM), resulted in a clear redistribution where the majority of nematodes were localized within the MZ (Figure 7c). This demonstrates that myristic acid exerts a repellent effect on the chemotactic migration of J2 nematodes.

Hexadecanal exhibited a similar trend on the chemotactic migration of M. incognita second-stage juveniles (J2) as myristic acid, albeit with a moderately weaker efficacy. In the MZ following metabolite application, nematodes accounted for 60.3% of the total population at the highest hexadecanal concentration tested (4 mM). Under identical myristic acid concentration gradients, however, the proportion of nematodes in the MZ reached 68.3% (Figure 7d). This comparative analysis indicates that myristic acid exerts a stronger repellent effect on J2 nematodes than hexadecanal.

Based on the importance of Mi-flp-1 and Mi-flp-18 during the chemotaxis of M. incognita to host plant roots [10]. To further investigate the effects of two metabolites observed in the one-choice attraction assay, second-stage juveniles (J2) of M. incognita were recovered from the assay plates and subjected to analysis of expression changes in the Mi-flp-1 and Mi-flp-18 genes. In the present study, both metabolites significantly downregulated the expression levels of both genes. Notably, myristic acid exhibited a more pronounced suppressive effect compared to hexadecanal. Regarding the two distinct Mi-flp genes, both root-derived metabolites exerted a disproportionately greater suppressive effect on Mi-flp-1 expression compared to Mi-flp-18. Notably, myristic acid significantly downregulated Mi-flp-1 transcript levels even at low treatment concentrations. This potent suppression of Mi-flp-1 may constitute a key underlying mechanism for the observed loss of chemotactic migration function in M. incognita J2. Together, these results confirmed the capacity of candidate root-derived metabolites to disrupt M. incognita chemotaxis towards cucumber root.

4. Discussion

Our study demonstrates that M. incognita infection triggers extensive metabolic reprogramming in cucumber roots, characterized by accumulation of lipids, organic acids, and the enrichment of defense-related pathways including alkaloid biosynthesis and linoleic acid metabolism (Figure 2, Figure 3 and Figure 4). This aligns with established plant defense mechanisms against biotic stress [27,28,29]. Plants have developed different defense systems against invading pests and pathogens, relying on the coordinated action of multiple signaling pathways [30,31]. Deciphering how plants interact with pathogens offers substantial promise for reducing losses in both agricultural yield and crop quality, contributing to improved farming outcomes. In susceptible host plants of M. incognita, dramatic morphological changes occur in host roots, which also leads to a diverse metabolic process in the roots [32,33]. In this study, linoleic acid metabolism was strongly affected by M. incognita infection in cucumber roots (Figure 3). This pattern is frequently observed in plant immune systems [34,35]. The pathway of Tropane, piperidine, and pyridine alkaloid biosynthesis was also enriched after M. incognita parasitism. Alkaloids including tropane and piperidine derivatives may disrupt nematode neuromuscular signaling [36], while linoleic acid metabolism potentially enhances membrane integrity and jasmonate-mediated defense signaling. Notably, lipid metabolism serves as an essential biochemical pathway participating in plant defense mechanisms [37]. Utilizing untargeted metabolomics, this study demonstrates that M. incognita infection significantly alters the root metabolome of cucumber.

Key enriched metabolic pathways include alkaloid biosynthesis (tropane, piperidine, and pyridine alkaloids), linoleic acid metabolism, and butanoate metabolism. It has been established that alkaloid biosynthesis pathways are associated with plant responses to both biotic and abiotic stresses [38]. Linoleic acid metabolism, a branch of fatty acid metabolism, also serves as a critical defense mechanism when activated under biotic stress [39]. Differential metabolite analysis in this study further revealed that lipids and lipid-like molecules exhibited the most substantial alterations in relative abundance. These findings collectively indicate that M. incognita infection induces immune responses in cucumber, manifested through metabolomic reprogramming.

Under biotic stress, plants can modify their metabolic profiles as a defense mechanism, producing various secondary metabolites to enhance stress resistance [40]. This is in line with findings in Arabidopsis roots infected by Heterodera schachtii, where significant metabolomic alterations were documented at 15 dpi (days post inoculation) [29], and in tomato-M. incognita compatible interactions showing dynamic transcriptomic/metabolomic shifts during giant cell formation and maintenance (days to weeks post-infection) [17]. While earlier timepoints might reveal initial defense signaling events, our sampling at 14 DPI captured significant metabolic reprogramming associated with the established parasitic phase, including the production of metabolites capable of directly interfering with nematode physiology and behavior. Notably, the upregulation of lipid-like metabolites including myristic acid and hexadecanal (

Table 1. Screening of candidate metabolites in root tissues of cucumber.

Metabolites	log2(FC)	KEGG ID	Pathway	VIP	p-Value
Myristic acid	0.66444	C06424	Fatty acid biosynthesis	1.78423	0.02618
Hexadecanal	1.32102	C00517	Fatty acid degradation	1.15038	0.00049

) suggests the potential role in response to the M. incognita parasitism. Alterations in the metabolome constitute the biochemical basis for plant responses to environmental stimuli. Given that M. incognita reprograms host metabolism to establish feeding sites [23,32], the gall microenvironment likely induces localized accumulation of defense metabolites. Lipids like myristic acid and hexadecanal may be synthesized de novo in giant cells or adjacent vascular tissues to directly impair nematode development. Experimental validation confirmed that all the two compounds exerted dose-dependent inhibitory effects on M. incognita egg hatching, juvenile mortality, and root infectivity. Notably, concentrations ≥ 1 mM induced significant suppression. However, it is critical to acknowledge that these effective in vitro concentrations (≥1 mM) likely exceed physiologically relevant levels within cucumber root tissues or the rhizosphere. This represents an important limitation, as we did not quantify absolute metabolite concentrations in vivo during infection. Nevertheless, the observed bioactivity mechanistically validates their nematode-suppressive potential.

Previous studies indicate that root exudates influence nematode egg hatching [41]. As these plant-derived metabolites are induced by M. incognita, they likely play pivotal roles in cucumber’s defense against nematode infection. Myristic acid is a member of the fatty acid family. Related research showed that different fatty acids, including palmitic acid and linoleic acid, could reduce M. incognita infection of tomato [42]. Current research on myristic acid in plants has primarily focused on its role in abiotic stress responses. For example, studies show that drought stress triggers the accumulation of myristic acid [43]. Research about tomato root exudates infected by M. incognita showed that tomato could secrete specific metabolites include myristic acid to improve the colonization of nematocidal Proteus vulgaris [44]. Another study of tomato showed that myristic acid were reduced in tomato leaves under root-knot nematode stress, which showed the effects of root-knot nematode on fatty acids [16]. Studies have shown that hexadecanal can be produced in insects infected by entomopathogenic nematodes, serving as an insect repellent and being applicable to the biocontrol of M. incognita [45,46]. Plant-derived metabolites may suppress root-knot nematode disease by inhibiting infective capability. In this study, all two metabolites impaired nematode infectivity, ultimately reducing root gall formation in cucumber. Research has identified nematode-suppressive effects in compounds such as amino acids (arginine and lysine), organic acids (vanillic acid, tannic acid, lauric acid), and flavonoids, which could regulate plant growth [26,47,48]. Notably, myristic acid from this study belong to the lipids and lipid-like molecules. Direct application of these plant-derived metabolites to the cucumber rhizosphere significantly reduced root gall numbers, suggesting their potential to regulate nematode–host interactions and control root-knot nematode disease. On the other hand, nematode infection significantly reduced several metabolites that are involved in immunity, including pipecolic acid (Table S1), and it has potential use in root-knot nematode management [49,50].

Furthermore, we determined the effects of two metabolites on the chemotaxis of M. incognita J2s, which determines whether nematodes can complete the infection of cucumber roots [51,52]. The results show that these two root-derived metabolites disrupted the movement of M. incognita to cucumber roots (Figure 7). The disruption of nematode chemotaxis plays a key role in suppressing RKN disease in plant metabolites–M. incognita interaction [4,26]. Several previous studies have shown that a variety of metabolites are capable of repelling RKNs, such as fatty acids, terpenoids, and phenolic acids including myristic acids [53]. In the present study, the pH of metabolite treatments may represent an external factor influencing the motility of M. incognita second-stage juveniles (J2). Previous studies have demonstrated that pH gradients can independently modulate nematode migratory behavior [54]. Thus, future experiments should rigorously control for pH effects to isolate the specific bioactivity of the metabolites.

Moreover, the M. incognita J2s separated from the one-choice assay under different differentially accumulated metabolites treatments showed a significant decrease in the transcript levels of Mi-flp-1 and Mi-flp-18 as compared to cucumber root tips (Figure 7). These findings are in agreement with previous studies confirming the importance of Mi-flp-1 and Mi-flp-18 in regulating the chemotaxis and infection of M. incognita [10]. The chemotaxis of M. incognita towards host plants is orchestrated by a sophisticated neuropeptide signaling system, which enables RKNs to perceive and interpret host-derived chemical cues. Among these neuropeptides, Mi-flp-1 and Mi-flp-18 (FMRFamide-like peptides) play pivotal roles in modulating nematode sensory and locomotor behaviors during host-seeking migration [8,10,55]. In this study, two metabolites reduced the transcript levels of Mi-flp-1 and Mi-flp-18 in M. incognita even at low concentrations (Figure 7). The results further demonstrating the potential of these two metabolites to disrupt M. incognita chemotaxis and infection.

5. Conclusions

In conclusion, alterations in the root metabolome play a critical role in the host’s response to root-knot nematode infection. In this study, M. incognita infection triggered significant modifications in the cucumber root metabolome, characterized by increased relative abundance of lipids and lipid-like molecules and organic acids, alongside enrichment of multiple metabolic pathways associated with biotic stress responses. Furthermore, two differentially accumulated metabolites include myristic acid and hexadecanal were demonstrated the inhibitory activity against M. incognita infectivity. In addition, both myristic acid and hexadecanal reduced the expression of Mi-flp-1 and Mi-flp-18 in M. incognita even at low concentrations further demonstrating the capacity of these two metabolites to disrupt M. incognita chemotaxis. These findings provide a scientific foundation for developing ecological control strategies against root-knot nematode diseases. Future studies should establish dose–response relationships under physiologically relevant concentrations within cucumber root tissues and the rhizosphere, which is essential for translating these findings into field-applicable and sustainable nematode management strategies.