BACKGROUND:The efficacy of entomopathogenic nematodes (EPNs) in the biological control of insect pests can be influenced by the host's chemical defenses. Phyllotreta flea beetles, among the most destructive pests of Brassica crops, deploy highly reactive glucosinolate hydrolysis products as a defense against natural enemies. Here, we investigate the susceptibility of EPNs and their symbiotic bacteria to glucosinolate hydrolysis products and assess how this defense shapes the interaction between the horseradish flea beetle, Phyllotreta armoraciae, and EPNs. RESULTS:Glucosinolate hydrolysis products were detected in uninjured P. armoraciae larvae but not in adults, and their levels were unaffected by EPN infection. EPNs and their bacterial symbionts were susceptible to glucosinolate hydrolysis products in vitro, with EPN immotility rates ranging from 35% to 96% and bacterial growth suppression from 20% to 85% at biologically relevant concentrations. However, reducing the levels of glucosinolate hydrolysis products in larvae, either by silencing myrosinase gene expression or by feeding on different Arabidopsis genotypes, did not make them more susceptible to EPNs. Nevertheless, the food plant influenced larval susceptibility to EPNs and the relative abundance of EPN bacterial symbionts in infected larvae. CONCLUSION:Although glucosinolate hydrolysis products are toxic to EPNs and their symbiotic bacteria, they did not protect P. armoraciae larvae from EPN infection. However, the larval food plant influenced EPN susceptibility and bacterial community composition, highlighting the role of host plant traits in shaping insect-EPN interactions. These findings provide new insights into the limitations of EPN-based biocontrol against glucosinolate-sequestering pests. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Aboveground induction of plant defense pathways can shape root-associated microbial communities. However, whether these changes are pathway-specific and how they affect plant growth and resistance remains unclear. We evaluated how induction of the Jasmonic Acid (JA) and Salicylic Acid (SA) defense pathways shapes the root microbiome of Brassica oleracea, and whether these soil-mediated shifts affect plant growth and resistance to herbivory in a subsequent generation using a plant-soil feedback (PSF) approach. In the conditioning phase, defense pathways were induced either through foliar application of methyl jasmonate (MeJA) and SA solutions, or through herbivory by caterpillars (JA) and aphids (SA). Both pathways led to distinct shifts in microbial communities, with bacterial and fungal composition varying by pathway identity and induction method. JA induction resulted in more differentially abundant ASVs than SA, particularly with Proteobacteria depletion. Conversely, Planctomycetota (bacteria) and Mortierellomycota (fungi) were enriched under both pathways, suggesting that these represent general stress-responsive groups. In the feedback phase, JA- and SA-conditioned soils had no effect on resistance under high aphid pressure, whereas under low aphid density, plants grown in SA-conditioned soil exhibited reduced phloem feeding and lower aphid population development. Together, our results indicate that benefits provided by the defense-shaped root microbiome depend on pest pressure intensity and arise from overall community shifts rather than specific taxa enrichment. Our findings underscore the complex interactions between plant-defense pathways, rhizosphere microbes, and herbivores.

Abstract Changes in intracellular calcium ion (Ca²⁺) concentrations generate characteristic signatures that are decoded by specialized Ca²⁺-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca²⁺ at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca²⁺-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca²⁺ sensor that couples Ca²⁺ perception to JA-mediated defense and abiotic stress responses in plants.

Fungi readily colonize the inner bark (phloem) of spruce and other conifers despite these tissues having a high concentration of antifungal defense metabolites. These compounds include stilbenes, flavonoids and other phenolic substances, mostly present as glucosides. Yet the underlying biochemical mechanisms by which fungi resist conifer phenolics remain largely unresolved. Using untargeted metabolomics, structural elucidation and biological and biochemical assays, we investigated how and why fungi metabolize the major phenolics of Norway spruce (Picea abies). Various fungi, including those associated with bark beetles, were found to hydrolyze stilbene glucosides to their corresponding aglucones. Two basidiomycetes, the saprotroph Coprinellus radians and Cylindrobasidium ipidophilum, a symbiont of the Eurasian spruce bark beetle Ips typographus, then converted the stilbene aglucones into α-ribofuranosylated derivatives, revealing a previously unrecognized pathway in tree-colonizing fungi. Ribosylation markedly reduced the antifungal activity of the aglucones and stabilized them against hydrolysis by fungal and I. typographus enzymes, preventing regeneration of the toxic aglucones. Ribosylation was also correlated with increased growth on spruce bark-containing medium. Hence, the ability to overcome major conifer bark defenses by conversion of toxic stilbene aglucones to non-toxic α-ribosides may explain the successful colonization of this tissue by fungi, some of which support I. typographus attack.

Conifers are a challenging host for herbivores since their tissues are very low in essential nutrients but high in chemical defenses. For herbivorous insects, such as phloem-colonizing bark beetles, mutualistic fungi may improve their diet by providing a nutritious mycelium. A recent study revealed that two filamentous fungi are mutualists of the European fir engraver beetle Pityokteines vorontzowi, but a potential nutritional contribution of the fungi, as well as their capability to degrade plant antiherbivore defenses remains unknown. We analyzed the nutrient content of the fungal mutualists Ophiostoma piceae and Geosmithia sp. F1 and examined their ability to degrade the constitutive chemical defenses of silver fir phloem in comparison to other fungi. Both mutualists turned out to be rich in amino acids, sugars, and B vitamins and were found to efficiently deplete their phloem media of several defenses. Strikingly, O. piceae not only accumulated the highest amounts of the B vitamin nicotinic acid of the 17 tested fungi but also showed a high ability to deplete its medium of chemical defenses, similar to the behavior of the Ips typographus mutualist Endoconidiophora polonica. Beetle-vectored, non-mutualistic fungi isolated from P. vorontzowi showed similar capacities to deplete defensive compounds, whereas non-fir-associated fungi were less effective in reducing their concentrations in the phloem medium. The nutritious mycelium of O. piceae and Geosmithia sp. F1 and the ability of these fungi to deplete the medium of major fir defense compounds likely facilitates the colonization of silver fir phloem by P. vorontzowi.

Plants produce a plethora of specialized metabolites that often play important roles in their defence against pathogenic microbes or herbivorous insects. Exposure of leaf-colonizing microbes to these metabolites influences their growth, and we hypothesize that it also has consequences for microbe-microbe interactions. In Brassicaceae plants like the model plant Arabidopsis thaliana, glucosinolates and their biologically active derivatives, the isothiocyanates, are major defence metabolites. Adapted plant pathogens like Pseudomonas spp. use the hydrolase SaxA to convert the antimicrobial isothiocyanate sulforaphane to a non-toxic amine, whereas non-adapted commensal microbes are inhibited by this plant toxin. We used Plantibacter sp. 2H11-2 as a model commensal in co-culture with either Pseudomonas viridiflava 3D9 wildtype or a saxA-knock-out mutant. Both strains were isolated from the same wild A. thaliana population. Without isothiocyanate, Plantibacter grew better alone than with Pseudomonas, a potential competitor. At high isothiocyanate concentrations, however, the commensal was dependent on SaxA-mediated isothiocyanate degradation in both solid and liquid medium. At intermediate isothiocyanate concentrations, Plantibacter's transcriptome changed in response to sulforaphane in monoculture but not in co-culture with Pseudomonas, suggesting that it was fully protected from this toxin. In return, Plantibacter caused transcriptional changes in Pseudomonas, suppressing biofilm formation and increasing amino acid metabolism gene expression which might suppress virulence and so contribute to plant health. Together, we find that degradation of an antimicrobial plant metabolite can protect a commensal to depend on a pathogen-produced virulence factor, suggesting effects on community composition in environments where microbes are exposed to ITCs.

Fungal endophytes of grasses and other herbaceous plants have been known to provide plants with anti-herbivore defence compounds, but there is little information about whether the endophytes of trees also engage in such mutualisms. We investigated the influence of the endophytic fungus Cladosporium sp. on the chemical defences of black poplar (Populus nigra) trees and the consequences for feeding preference and fitness of herbivorous insects and insect community assembly. Endophyte colonisation increased both constitutive- and induced poplar defences. Generalist Lymantria dispar larvae preferred and performed better on uninfected over endophyte-infected poplar leaves, most likely due to higher concentrations of salicinoids in endophyte-inoculated leaves and the endophyte-produced alkaloid stachydrine. Under field conditions, the endophytic fungus shapes insect community assembly i. a. attracting aphids, which can excrete stachydrine. Our results show that endophytic fungi play a crucial role in the defence against insects from different feeding guilds and thereby structuring insect communities.

After consumption by herbivores, plant antimicrobial defense compounds may enhance herbivore immunity to pathogenic microbes. In conifer-bark beetle interactions, beetles ingest large quantities of phloem tissue containing high concentrations of antimicrobial phenolic glucosides, such as stilbenes and flavonoids. It is not known, however, if these compounds increase bark beetle resistance to pathogens. We showed that Eurasian spruce bark beetles (Ips typographus) attacking Norway spruce (Picea abies) hydrolyze phenolic glucosides to their corresponding aglucones increasing their antifungal activity. However, the entomopathogen Beauveria bassiana, a natural fungal parasite of these beetles, detoxifies stilbene and flavonoid aglucones by forming methylglucoside derivatives. A two-step pathway involving a UDP-glycosyltransferase and an O-methyltransferase produces phenolic O-methylglucosides that are no longer toxic to B. bassiana and are stable to β-glucosidase action. Compared to wild-type strains of B. bassiana, mutant strains knocked out in the genes of this pathway exhibited decreased methylglucoside formation, slower growth on medium containing phenolic compounds, and reduced virulence toward bark beetles. Hence, methylglucosylation of plant-derived phenolics is a detoxification process that significantly increases the ability of B. bassiana to parasitize host insects consuming plant tissue high in phenolics, such as conifer phloem. This is one of the few examples of an entomopathogen that is able to resist the plant-derived defenses of an insect host.

Interspecific hybridization has influenced plant evolution and diversification. However, how hybridization may affect metabolic diversity, especially in naturally occurring hybridization zones, is unclear. In this study, we selected a Baccharis (Asteraceae) hybrid complex consisting of B. linearis, B. macraei, and B. × intermedia and characterized its metabolic profiles in multiple hybridization zones in central Chile to determine how hybridization affects plant chemistry. Untargeted liquid chromatography-time of flight mass spectrometry analysis of a total of 411 plant individuals collected in the field revealed that the hybrid B. × intermedia combines the metabolic profiles of its two parental species, B. linearis and B. macraei, independent of season, location, and environment. This combinatorial effect was observed in the specialized metabolites, while the primary metabolism did not differ between species. The metabolic diversity of the hybrid exceeded that of the parental species and was influenced by latitude, with higher metabolic diversity in the northern populations than in those in the south. In summary, our results demonstrate that natural interspecific hybridization can quickly increase the diversity of specialized metabolites. This could enhance protection against biotic or abiotic stressors, particularly in changing environmental conditions.

Brassicales plants defend themselves with glucosinolates that, upon herbivory, are hydrolyzed into toxic isothiocyanates (ITCs) and other derivatives. The side chain diversity of glucosinolates results in a range of structurally distinct products, but how this chemical variation affects herbivores and their detoxification responses remains incompletely understood. Here, we show the effects of ITC hydrolysis products with various side chains on Spodoptera littoralis larvae and their detoxification system. ITCs inhibit larval growth to varying degrees, depending on the chemical nature of their side chain. The larvae metabolize ITCs by conjugating them to glutathione in the mercapturic acid pathway and to lysine forming an amine conjugate. Over half of the 34 S. littoralis glutathione-S-transferases (GSTs), tested as His-tagged derivatives, actively conjugate ITCs, with most catalyzing reactions with multiple substrates. Larval performance on various ITC-containing diets correlates positively with GST activity, highlighting this detoxification system's role in supporting growth on glucosinolate-containing plants. The propensity of multiple GSTs to react with an individual ITC and the wide expression of GST-encoding genes across larval organs likely promote the ability of this generalist herbivore to thrive on glucosinolate-defended Brassicales plants. These findings provide insight into herbivore adaptation and may inform future research on plant-insect interactions.

Plants are challenged regularly with multiple types of biotic stress factors, such as pathogens or insect herbivores, in their environment. To detect and defend against pathogens, plants have evolved an innate immune system in which intracellular receptors in the so-called effector-triggered immunity play a vital role. In Arabidopsis thaliana the Toll/interleukin-1 receptors (TIRs) domain is related to intracellular immunity receptors, for example in TIR-NBS-LRR (TNL) proteins. Among the TIR domain carrying proteins, very little is known about the function of the TIR-X proteins. Here, we focus on the recently described TIR-X (TIRP; At5g44900) to analyze its role in phytohormone-mediated plant defense through gene expression and phytohormone quantification. Therefore, we employed two fungal pathogens, the necrotrophic Alternaria brassicicola and the hemibiotrophic Verticillium dahliae, to infect A. thaliana WT (Col-0), TIRP knock-out, and TIRP overexpressing lines for comparative analyses. Furthermore, we included the insect herbivore Spodoptera littoralis and a treatment with S. littoralis egg extract on the plants to analyze any role of TIRP during these attacks. We found that both A. brassicicola and V. dahliae infections increased TIRP gene expression systemically. The salicylic acid content was higher in the TIRP overexpressing line, corresponding to a better S. littoralis larval growth performance in feeding assays. However, since we never observed clear infection-related differences in jasmonate or salicylic acid levels between the wild type and the two transgenic Arabidopsis lines, our results rule out the possibility that TIRP acts via the regulation of phytohormone synthesis and accumulation.

Thyme species, including Thymus vulgaris, T. kotschyanus (drought-tolerant) and T. serpyllum (drought-sensitive), are valuable medicinal herbs. They are often grown in arid regions and are increasingly suffering from water stress due to climate change. Here, we analyzed the metabolome and expression of selected genes in leaves of these species under drought stress with and without treatment with the phytohormone abscisic acid (ABA). Among the terpenes, dominant metabolites in thyme, thymol was the most important terpenoid component, followed by thymoquinone, carvacrol and p-cymene in all three species. Drought stress reduced terpene concentrations, while moderate ABA levels increased them. T. kotschyanus showed the highest concentrations of thymol and carvacrol after combined treatment with drought and ABA. Metabolite accumulation was partially correlated with genes related to terpenoid biosynthesis. The combined treatment of drought stress and ABA resulted in a significant reduction of the stress hormone jasmonic acid and an increase of its biosynthetic precursor, OPDA (cis-12-oxophytodienoic acid), in all species. The present research results indicate that ABA treatment at moderate concentrations could be used as a measure to increase the production of some pharmaceutically active phenolic monoterpenes in T. vulgaris, T. serpyllum and T. kotschyanus and increase the stress resistance of the plants.

High soil salinity affects plant growth, yield, and water use efficiency, leading to drought and ion toxicity. Silicon (Si), a crucial element in soil, can mitigate such stress. Si neutralizes harmful impacts, reduces Na+ uptake, and promotes plant growth. It benefits higher plants like grasses and cultivated crops. However, its role in maize cultivars is rarely reported. The present study aimed to evaluate the impact of exogenous Si application on maize plant growth, physiology, gene activation, and phytohormonal regulation under salinity stress. Therefore, a hydroponic experiment was conducted to study the impact of salt (100 mM NaCl) on two different maize varieties, the salt-sensitive Jalal and the salt-tolerant Iqbal, along with and without Si enrichment in pots or Si foliar spray. Our findings revealed that various phenotypical growth parameters as well as physiological parameters were significantly affected due to salt stress. However, the presence of Si mitigated the stress responses in both varieties. Moreover, we found that Si application reduced the NaCl-induced effects on abscisic acid and jasmonates in both varieties. Based on our findings, we concluded that Si application may lead to a reduction of NaCl-mediated salt stress on maize plants and help the plant to grow better.

Herbivorous insects have evolved many fascinating adaptations to overcome the chemical defenses of their host plants. This study employed targeted and untargeted metabolomics, coupled with isotope labeling, to shed light on the metabolism of salicinoids-potent antiherbivore phenolic defenses present in the Salicaceae family-in the poplar-specialized leaf beetle, Chrysomela tremulae. C. tremulae was found to produce a range of metabolites from salicortin and utilize the essential amino acid tryptophan and its breakdown products, namely kynurenine, kynurenic acid, and 4-hydroxyquinoline, to form novel conjugates with the salicinoid metabolite saligenin, which are then excreted in the feces of the beetles. Saligenin and its conjugates are not toxic to C. tremulae and similar metabolic pathways were found in other poplar herbivores. Experimental analyses of the gut microbiota revealed that there is no microbial contribution to the formation of tryptophan metabolite-saligenin conjugates. The production of such substances by insect herbivores may be a critical adaptation that enables specialists to survive on a diet high in salicinoid defense compounds. Therefore, identifying the underlying detoxification mechanisms creates opportunities to develop targeted anti-insect agents for protecting salicaceous trees.

Progestogens and androgens have been found in many plants, but little is known about their physiological function. We used a previously established UPLC-ESI-MS/MS method to analyze progestogen and androgen profiles in fungal infections. Here we show that dehydroepiandrosterone (DHEA), a C19 steroid, specifically accumulates in shoots of Arabidopsis thaliana (L.) HEYNH. infected with Alternaria brassicicola (SCHWEIN.) WILTSHIRE. Elevated DHEA levels in plants seem not to be product of fungal sterol/steroid precursor activity, but an intrinsic plant response to the infection. DHEA was applied exogenously to analyze the effects of the androgen on development and gene expression in A. thaliana. Our findings reveal that DHEA treatment downregulates membrane-associated, salicylic acid and abscisic acid-regulated, as well as stress-responsive genes. Notably, DHEA does not inhibit the isoprenoid or post-lanosterol pathway of the ergosterol biosynthesis. Moreover, A. brassicicola was also treated with DHEA to analyze the growth, sterol pattern and membrane-integrity. Our data suggest that DHEA enhances the permeability of plant and fungal biomembranes. We propose that DHEA accumulation is a plant defense response which reduces fungal growth in plant tissues.

Date palm (Phoenix dactylifera L.) is an important crop in arid regions and it is well adapted to desert ecosystems. To understand its remarkable ability to grow and yield in water-limited environments, we conducted experiments in which water was withheld for up to 4 weeks. In response to drought, root, rather than leaf, osmotic strength increased, with organic solutes such as sugars and amino acids contributing more to the osmolyte increase than minerals. Consistently, carbon and amino acid metabolism was acclimated toward biosynthesis at both the transcriptional and translational levels. In leaves, a remodeling of membrane systems was observed, suggesting changes in thylakoid lipid composition which, together with the restructuring of the photosynthetic apparatus, indicated an acclimation preventing oxidative damage. Thus, xerophilic date palm avoids oxidative damage under drought by combined prevention and rapid detoxification of oxygen radicals. Although minerals were expected to serve as cheap key osmotics, date palm also relies on organic osmolytes for osmotic adjustment in the roots during early drought acclimation. The diversion of these resources away from growth is consistent with the date palm strategy of generally slow growth in harsh environments and clearly indicates a trade-off between growth and stress-related physiological responses.

Insect oviposition success depends on selecting optimal host plants, guided by plant chemical cues critical for larval fitness. Yet, the specific metabolites shaping egg-laying choices remain unclear, as some enhance oviposition but inconsistently affect larval performance. Since larval success is wholly contingent on adult oviposition decisions, plant metabolites mediating both egg-laying behavior and larval fitness are pivotal to understand insect behavioral ecology and targeted pest control interventions. Using Brassica specialist, Plutella xylostella, we tested the impact of plant chemical defences on oviposition and larval fitness. We used eight varieties of Brassica plants to evaluate insect oviposition preference and subsequent larval fitness. Glucosinolates, key secondary metabolites of Brassica species influenced oviposition. Among these, 4-hydroxy-indol-3-ylmethylglucosinolate (4 H-I3M) was identified to impact oviposition, larval cellular immunity and survival against entomopathogens. Larvae reared on artificial diet containing 4 H-I3M also showed higher immunity and better survival against entomopathogens. Moreover, painting 4 H-I3M on plant cultivar lacking this compound or onto a paper disc, consistently induced oviposition behavior. This study demonstrates that (a) 4 H-I3M is both necessary and sufficient to induce oviposition (b) 4 H-I3M regulates larval cellular immunity and improves survival against entomopathogens. These results suggest that insects utilize plant compounds as proactive signals, guiding their choice of host plants to enhance larval immunity and ensure survival.

Wood-colonizing beetles are associated with a diversity of microbes many of which are thought to act as mutualists with their beetle hosts, but the evidence is usually anecdotal. The ship-timber beetle Elateroides dermestoides, one of the few fungus-farming but nonsocial ambrosia beetles, is described to have a mutualistic relationship with the yeast-like fungus Alloascoidea hylecoeti. Here, we tested the hypothesis that A. hylecoeti has a high nutrient content thus allowing it to function as a valuable food source for the solitary larvae of E. dermestoides, which bore into the wood of dead trees, an extremely nutrient-poor substrate. Our analyses revealed that A. hylecoeti is rich in soluble sugars, free amino acids, ergosterol, phosphorus, and potassium compared to the other fungi measured, and also accumulates high amounts of fatty acids, B vitamins and nitrogen. We also tested whether A. hylecoeti possesses chemical mechanisms to suppress antagonistic microbes. Extracts from A. hylecoeti and chemical compounds produced or accumulated by this fungus were found to significantly inhibit the growth of potentially competing fungi. The active substances include fungal-produced monoterpenes and acetic acid, as well as phenolic compounds accumulated from host tree tissues. Moreover, sufficient acetic acid was released by A. hylecoeti to drop the medium pH to as low as 3.6, which inhibited all tested competitors, whereas the growth of A. hylecoeti was promoted. Taken together, the nutritional properties and competitive ability of A. hylecoeti may make a major contribution to the success of its insect partner, the ship-timber beetle under natural conditions.

Highlight statement Osmotic strength of date palm roots increases with soil desiccation, for which the accumulation of organic osmolytes, such as sugars, is essential in complement to energetically cheap mineral osmotics. Date palm ( Phoenix dactylifera L.) is an important crop in arid regions that is well-adapted to desert ecosystems. To understand the remarkable ability to grow and yield in water-limited environments, experiments were conducted in a simulated desert environment with water-withholding for up to four weeks. In response to drought, root, rather than leaf, osmotic strength increased, with sugars contributing more to the osmolyte increase than minerals. Consistently, carbon and amino acid metabolism was acclimated toward biosynthesis at both the transcriptional and translational levels. In leaves, a remodeling of membrane systems was observed, suggesting changes in thylakoid lipid composition, which together with the restructuring of the photosynthetic apparatus, indicated an acclimation preventing oxidative damage. Thus, xerophilic date palm avoids oxidative damage under drought by combined prevention and rapid detoxification of oxygen radicals. Although minerals were expected to serve as cheap key osmotics, date palm also relies on organic osmolytes for osmotic adjustment of the roots during desiccation. The diversion of these resources away from growth is consistent with date palm’s strategy of generally slow growth in harsh environments and clearly indicates a trade-off between growth and stress-related physiological responses. ### Competing Interest Statement The authors have declared no competing interest.

Plasticity in plant traits, including secondary metabolites, is critical to plant survival and competitiveness under stressful conditions. The ability of a plant to respond effectively to combined stressors can be impacted by crosstalk in biochemical pathways, resource availability and evolutionary history, but such responses remain underexplored. In particular, we know little about intraspecific variation in response to combined stressors or whether such variation is associated with the stress history of a given population.Here, we investigated the consequences of combined water and herbivory stress for plant traits, including relative growth rate, leaf morphology and various measures of phytochemistry, using a common garden of Asclepias fascicularis milkweeds. To examine how plant trait means and plasticities depend on the history of environmental stress, seeds for the experiment were collected from across a gradient of aridity in the Great Basin, United States. We then conducted a factorial experiment crossing water limitation with herbivory.Plants responded to water limitation alone by increasing the evenness of UV-absorbent secondary metabolites and to herbivory alone by increasing the richness of metabolites. However, plants that experienced combined water and herbivory stress exhibited similar phytochemical diversity to well-watered control plants. This lack of plasticity in phytochemical diversity in plants experiencing combined stressors was associated with a reduction in relative growth rates.Leaf chemistry means and plasticities exhibited clinal variation corresponding to seed source water deficits. The total concentration of UV-absorbent metabolites decreased with increasing water availability among seed sources, driven by higher concentrations of flavonol glycosides, which are hypothesized to act as antioxidants, among plants from drier sites. Plants sourced from drier sites exhibited higher plasticity in flavonol glycoside concentrations in response to water limitation, which increased phytochemical evenness, but simultaneous herbivory dampened plant responses to water limitation irrespective of seed source.Synthesis. These results suggest that climatic history can affect intraspecific phytochemical plasticity, which may confer tolerance to water limitation, but that co-occurring herbivory disrupts such patterns. Global change is increasing the frequency and intensity of stress combinations, such that understanding intraspecific responses to combined stressors is critical for predicting the persistence of plant populations. These results suggest that climatic history can affect intraspecific phytochemical plasticity, which may confer tolerance to water limitation, but that co-occurring herbivory disrupts such patterns. Global change is increasing the frequency and intensity of stress combinations, such that understanding intraspecific responses to combined stressors is critical for predicting the persistence of plant populations.image