Structural diversity, hypothetical biosynthesis, chemical synthesis, and biological activity of Ganoderma meroterpenoids.

Covering: 2018 to 2022Meroterpenoids found in fungal species of the genus Ganoderma and known as Ganoderma meroterpenoids (GMs) are substances composed of a 1,2,4-trisubstituted benzene and a polyunsaturated side chain. These substances have attracted the attention of chemists and pharmacologists due to their diverse structures and significant bioactivity. In this review, we present the structures and possible biosynthesis of representative GMs newly found from 2018 to 2022, as well as chemical synthesis and biological activity of some interesting GMs. We propose for the first time a plausible biosynthetic pathway for GMs, which will certainly motivate further research on the biosynthetic pathway in Ganoderma species, as well as on chemical synthesis of GMs as important bioactive compounds for the purpose of drug development.

Inter-laboratory comparison of plant volatile analyses in the light of intra-specific chemodiversity.

INTRODUCTION: Assessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses. OBJECTIVES: In a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps. METHODS: Monoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures. RESULTS: Chemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles. CONCLUSION: Our ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.

Poplar protease inhibitor expression differs in an herbivore specific manner.

BACKGROUND: Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. RESULTS: We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. CONCLUSIONS: We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

The Occurrence of Sulfated Salicinoids in Poplar and Their Formation by Sulfotransferase1.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

Nutrient status not secondary metabolites drives herbivory and pathogen infestation across differently mycorrhized tree monocultures and mixtures.

Research aimed at understanding the mechanisms underlying the relationship between tree diversity and antagonist infestation is often neglecting resource-use complementarity among plant species. We investigated the effects of tree species identity, species richness, and mycorrhizal type on leaf herbivory and pathogen infestation. We used a tree sapling experiment manipulating the two most common mycorrhizal types, arbuscular mycorrhiza and ectomycorrhiza, via respective tree species in monocultures and two-species mixtures. We visually assessed leaf herbivory and pathogen infestation rates, and measured concentrations of a suite of plant metabolites (amino acids, sugars, and phenolics), leaf elemental concentrations (carbon, nitrogen, and phosphorus), and tree biomass. Tree species and mycorrhizal richness had no significant effect on herbivory and pathogen infestation, whereas species identity and mycorrhizal type had. Damage rates were higher in arbuscular mycorrhizal (AM) than in ectomycorrhizal (EM) trees. Our structural equation model (SEM) indicated that elemental, but not metabolite concentrations, determined herbivory and pathogen infestation, suggesting that the investigated chemical defence strategies may not have been involved in the effects found in our study with tree saplings. Other chemical and physical defence strategies as well as species identity as its determinant may have played a more crucial role in the studied saplings. Furthermore, the SEM indicated a direct positive effect of AM trees on herbivory rates, suggesting that other dominant mechanisms, not considered here, were involved as well. We found differences in the attribution of elemental concentrations between the two rates. This points to the fact that herbivory and pathogen infestation are driven by distinct mechanisms. Our study highlights the importance of biotic contexts for understanding the mechanisms underlying the effects of biodiversity on tree-antagonist interactions.

Volatile emission and biosynthesis in endophytic fungi colonizing black poplar leaves.

Plant volatiles play a major role in plant-insect interactions as defense compounds or attractants for insect herbivores. Recent studies have shown that endophytic fungi are also able to produce volatiles and this raises the question of whether these fungal volatiles influence plant-insect interactions. Here, we qualitatively investigated the volatiles released from 13 endophytic fungal species isolated from leaves of mature black poplar (Populus nigra) trees. The volatile blends of these endophytes grown on agar medium consist of typical fungal compounds, including aliphatic alcohols, ketones and esters, the aromatic alcohol 2-phenylethanol and various sesquiterpenes. Some of the compounds were previously reported as constituents of the poplar volatile blend. For one endophyte, a species of Cladosporium, we isolated and characterized two sesquiterpene synthases that can produce a number of mono- and sesquiterpenes like (E)-ß-ocimene and (E)-ß-caryophyllene, compounds that are dominant components of the herbivore-induced volatile bouquet of black poplar trees. As several of the fungus-derived volatiles like 2-phenylethanol, 3-methyl-1-butanol and the sesquiterpene (E)-ß-caryophyllene, are known to play a role in direct and indirect plant defense, the emission of volatiles from endophytic microbial species should be considered in future studies investigating tree-insect interactions.

Herbivory meets fungivory: insect herbivores feed on plant pathogenic fungi for their own benefit.

Plants are regularly colonised by fungi and bacteria, but plant-inhabiting microbes are rarely considered in studies on plant-herbivore interactions. Here we show that young gypsy moth (Lymantria dispar) caterpillars prefer to feed on black poplar (Populus nigra) foliage infected by the rust fungus Melampsora larici-populina instead of uninfected control foliage, and selectively consume fungal spores. This consumption, also observed in a related lepidopteran species, is stimulated by the sugar alcohol mannitol, found in much higher concentration in fungal tissue and infected leaves than uninfected plant foliage. Gypsy moth larvae developed more rapidly on rust-infected leaves, which cannot be attributed to mannitol but rather to greater levels of total nitrogen, essential amino acids and B vitamins in fungal tissue and fungus-infected leaves. Herbivore consumption of fungi and other microbes may be much more widespread than commonly believed with important consequences for the ecology and evolution of plant-herbivore interactions.

Plant volatile emission depends on the species composition of the neighboring plant community.

BACKGROUND: Plants grow in multi-species communities rather than monocultures. Yet most studies on the emission of volatile organic compounds (VOCs) from plants in response to insect herbivore feeding focus on one plant species. Whether the presence and identity of neighboring plants or plant community attributes, such as plant species richness and plant species composition, affect the herbivore-induced VOC emission of a focal plant is poorly understood. METHODS: We established experimental plant communities in pots in the greenhouse where the focal plant species, red clover (Trifolium pratense), was grown in monoculture, in a two species mixture together with Geranium pratense or Dactylis glomerata, or in a mixture of all three species. We measured VOC emission of the focal plant and the entire plant community, with and without herbivory of Spodoptera littoralis caterpillars caged on one red clover individual within the communities. RESULTS: Herbivory increased VOC emission from red clover, and increasing plant species richness changed emissions of red clover and also from the entire plant community. Neighbor identity strongly affected red clover emission, with highest emission rates for plants growing together with D. glomerata. CONCLUSION: The results from this study indicate that the blend of VOCs perceived by host searching insects can be affected by plant-plant interactions.

Salicylic acid activates poplar defense against the biotrophic rust fungus Melampsora larici-populina via increased biosynthesis of catechin and proanthocyanidins.

Poplar trees synthesize flavan-3-ols (catechin and proanthocyanidins) as a defense against foliar rust fungi, but the regulation of this defense response is poorly understood. Here, we investigated the role of hormones in regulating flavan-3-ol accumulation in poplar during rust infection. We profiled levels of defense hormones, signaling genes, and flavan-3-ol metabolites in black poplar leaves at different stages of rust infection. Hormone levels were manipulated by external sprays, genetic engineering, and drought to reveal their role in rust fungal defenses. Levels of salicylic acid (SA), jasmonic acid, and abscisic acid increased in rust-infected leaves and activated downstream signaling, with SA levels correlating closely with those of flavan-3-ols. Pretreatment with the SA analog benzothiadiazole increased flavan-3-ol accumulation by activating the MYB-bHLH-WD40 complex and reduced rust proliferation. Furthermore, transgenic poplar lines overproducing SA exhibited higher amounts of flavan-3-ols constitutively via the same transcriptional activation mechanism. These findings suggest a strong association among SA, flavan-3-ol biosynthesis, and rust resistance in poplars. Abscisic acid also promoted poplar defense against rust infection, but likely through stomatal immunity independent of flavan-3-ols. Jasmonic acid did not confer any apparent defense responses to the fungal pathogen. We conclude that SA activates flavan-3-ol biosynthesis in poplar against rust infection.

What makes a volatile organic compound a reliable indicator of insect herbivory?

Plants that are subject to insect herbivory emit a blend of so-called herbivore-induced plant volatiles (HIPVs), of which only a few serve as cues for the carnivorous enemies to locate their host. We lack understanding which HIPVs are reliable indicators of insect herbivory. Here, we take a modelling approach to elucidate which physicochemical and physiological properties contribute to the information value of a HIPV. A leaf-level HIPV synthesis and emission model is developed and parameterized to poplar. Next, HIPV concentrations within the canopy are inferred as a function of dispersion, transport and chemical degradation of the compounds. We show that the ability of HIPVs to reveal herbivory varies from almost perfect to no better than chance and interacts with canopy conditions. Model predictions matched well with leaf-emission measurements and field and laboratory assays. The chemical class a compound belongs to predicted the signalling ability of a compound only to a minor extent, whereas compound characteristics such as its reaction rate with atmospheric oxidants, biosynthesis rate upon herbivory and volatility were much more important predictors. This study shows the power of merging fields of plant-insect interactions and atmospheric chemistry research to increase our understanding of the ecological significance of HIPVs.

Aboveground phytochemical responses to belowground herbivory in poplar trees and the consequence for leaf herbivore preference.

Belowground (BG) herbivory can influence aboveground (AG) herbivore performance and food preference via changes in plant chemistry. Most evidence for this phenomenon derives from studies in herbaceous plants but studies in woody plants are scarce. Here we investigated whether and how BG herbivory on black poplar (Populus nigra) trees by Melolontha melolontha larvae influences the feeding preference of Lymantria dispar (gypsy moth) caterpillars. In a food choice assay, caterpillars preferred to feed on leaves from trees that had experienced attack by BG herbivores. Therefore, we investigated the effect of BG herbivory on the phytochemical composition of P. nigra trees alone and in combination with AG feeding by L. dispar caterpillars. BG herbivory did not increase systemic AG tree defences like volatile organic compounds, protease inhibitors and salicinoids. Jasmonates and salicylic acid were also not induced by BG herbivory in leaves but abscisic acid concentrations drastically increased together with proline and few other amino acids. Leaf coating experiments with amino acids suggest that proline might be responsible for the caterpillar feeding preference via presumptive phagostimulatory properties. This study shows that BG herbivory in poplar can modify the feeding preference of AG herbivores via phytochemical changes as a consequence of root-to-shoot signaling.

Specificity of Herbivore Defense Responses in a Woody Plant, Black Poplar (Populus nigra).

The specificity of woody plant defense responses to different attacking herbivores is poorly known. We investigated the responses of black poplar (Populus nigra) to leaf feeding by three lepidopteran species (Lymantria dispar, Laothoe populi and Amata mogadorensis) and two leaf beetle species (Phratora vulgatissima and Chrysomela populi). Of the direct defenses monitored, increases in trypsin protease inhibitor activity and the salicinoid salicin were triggered by herbivore damage, but this was not herbivore-specific. Moreover, the majority of leaf salicinoid content was present constitutively and not induced by herbivory. On the other hand, volatile emission profiles did vary among herbivore species, especially between coleopterans and lepidopterans. Monoterpenes and sesquiterpenes were induced in damaged and adjacent undamaged leaves, while the emission of green leaf volatiles, aromatic and nitrogen-containing compounds (known to attract herbivore enemies) was restricted to damaged leaves. In conclusion, indirect defenses appear to show more specific responses to attacking herbivores than direct defenses in this woody plant.

Leaf rust infection reduces herbivore-induced volatile emission in black poplar and attracts a generalist herbivore.

Plants release complex volatile blends after separate attack by herbivores and pathogens, which play many roles in interactions with other organisms. Large perennials are often attacked by multiple enemies, but the effect of combined attacks on volatile emission is rarely studied, particularly in trees. We infested Populus nigra trees with a pathogen, the rust fungus Melampsora larici-populina, and Lymantria dispar caterpillars alone and in combination. We investigated poplar volatile emission and its regulation, as well as the behavior of the caterpillars towards volatiles from rust-infected and uninfected trees. Both the rust fungus and the caterpillars alone induced volatile emission from poplar trees. However, the herbivore-induced volatile emission was significantly reduced when trees were under combined attack by the herbivore and the fungus. Herbivory induced terpene synthase transcripts as well as jasmonate concentrations, but these increases were suppressed when the tree was additionally infected with rust. Caterpillars preferred volatiles from rust-infected over uninfected trees. Our results suggest a defense hormone crosstalk upon combined herbivore-pathogen attack in poplar trees which results in lowered emission of herbivore-induced volatiles. This influences the preference of herbivores, and might have other far-reaching consequences for the insect and pathogen communities in natural poplar forests.

Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications.

Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta-analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals.

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection.

Phenolic secondary metabolites are often thought to protect plants against attack by microbes, but their role in defense against pathogen infection in woody plants has not been investigated comprehensively. We studied the biosynthesis, occurrence, and antifungal activity of flavan-3-ols in black poplar (Populus nigra), which include both monomers, such as catechin, and oligomers, known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases and two anthocyanidin reductases from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis, leading to the formation of catechin [2,3-trans-(+)-flavan-3-ol] and epicatechin [2,3-cis-(-)-flavan-3-ol], respectively. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) accumulated higher amounts of catechin and PAs than uninfected trees. The de novo-synthesized catechin and PAs in the rust-infected poplar leaves accumulated significantly at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth. Poplar genotypes with constitutively higher levels of catechin and PAs as well as hybrid aspen (Populus tremula × Populus alba) overexpressing the MYB134 transcription factor were more resistant to rust infection. Silencing PnMYB134, on the other hand, decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective antifungal defenses in poplar against foliar rust infection.

Phytochemical variation in treetops: causes and consequences for tree-insect herbivore interactions.

The interaction of plants and their herbivorous opponents has shaped the evolution of an intricate network of defences and counter-defences for millions of years. The result is an astounding diversity of phytochemicals and plant strategies to fight and survive. Trees are specifically challenged to resist the plethora of abiotic and biotic stresses due to their dimension and longevity. Here, we review the recent literature on the consequences of phytochemical variation in trees on insect-tree-herbivore interactions. We discuss the importance of genotypic and phenotypic variation in tree defence against insects and suggest some molecular mechanisms that might bring about phytochemical diversity in crowns of individual trees.

Two herbivore-induced cytochrome P450 enzymes CYP79D6 and CYP79D7 catalyze the formation of volatile aldoximes involved in poplar defense.

Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus × canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom.

Feeding Experience Affects the Behavioral Response of Polyphagous Gypsy Moth Caterpillars to Herbivore-induced Poplar Volatiles.

Plant volatiles influence host selection of herbivorous insects. Since volatiles often vary in space and time, herbivores (especially polyphagous ones) may be able to use these compounds as cues to track variation in host plant quality based on their innate abilities and previous experience. We investigated the behavioral response of naïve (fed on artificial diet) and experienced (fed on poplar) gypsy moth (Lymantria dispar) caterpillars, a polyphagous species, towards constitutive and herbivore-induced black poplar (Populus nigra) volatiles at different stages of herbivore attack. In Y-tube olfactometer assays, both naïve and experienced caterpillars were attracted to constitutive volatiles and volatiles released after short-term herbivory (up to 6 hr). Naïve caterpillars also were attracted to volatiles released after longer-term herbivory (24-30 hr), but experienced caterpillars preferred the odor of undamaged foliage. A multivariate statistical analysis comparing the volatile emission of undamaged plants vs. plants after short and longer-term herbivory, suggested various compounds as being responsible for distinguishing between the odors of these plants. Ten compounds were selected for individual testing of caterpillar behavioral responses in a four-arm olfactometer. Naïve caterpillars spent more time in arms containing (Z)-3-hexenol and (Z)-3-hexenyl acetate than in solvent permeated arms, while avoiding benzyl cyanide and salicyl aldehyde. Experienced caterpillars avoided benzyl cyanide and preferred (Z)-3-hexenyl acetate and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) over solvent. Only responses to DMNT were significantly different when comparing experienced and naïve caterpillars. The results show that gypsy moth caterpillars display an innate behavioral response towards constitutive and herbivore-induced plant volatiles, but also that larval behavior is plastic and can be modulated by previous feeding experience.

Herbivore-induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar.

Numerous plant species emit volatile nitriles upon herbivory, but the biosynthesis as well as the relevance of these nitrogenous compounds in plant-insect interactions remains unknown. Populus trichocarpa has been shown to produce a complex blend of nitrogenous volatiles, including aldoximes and nitriles, after herbivore attack. The aldoximes were previously reported to be derived from amino acids by the action of cytochrome P450 enzymes of the CYP79 family. Here we show that nitriles are derived from aldoximes by another type of P450 enzyme in P. trichocarpa. First, feeding of deuterium-labeled phenylacetaldoxime to poplar leaves resulted in incorporation of the label into benzyl cyanide, demonstrating that poplar volatile nitriles are derived from aldoximes. Then two P450 enzymes, CYP71B40v3 and CYP71B41v2, were characterized that produce aliphatic and aromatic nitriles from their respective aldoxime precursors. Both possess typical P450 sequence motifs but do not require added NADPH or cytochrome P450 reductase for catalysis. Since both enzymes are expressed after feeding by gypsy moth caterpillars, they are likely to be involved in herbivore-induced volatile nitrile emission in P. trichocarpa. Olfactometer experiments showed that these volatile nitriles have a strong repellent activity against gypsy moth caterpillars, suggesting they play a role in induced direct defense against poplar herbivores.

Herbivore preference drives plant community composition.

Herbivores are important drivers of plant species coexistence and community assembly. However, detailed mechanistic information on how herbivores affect dominance hierarchies between plant species is scarce. Here, we used data of a multi-site herbivore exclusion experiment in grasslands to assess changes in the cover of 28 plant species in response to aboveground pesticide. application. Moreover, we assessed species-specific values of plant defense of these 28 species measured as the performance of a generalist caterpillar, and the preference of the caterpillar and a slug species in no-choice and choice feeding experiments, respectively. We show that more preferred species in the feeding experiments were those that increased in cover after herbivore exclusion in the field, whereas less preferred ones decreased. Herbivore performance and several measured leaf traits were not related to the change in plant cover in the field in response to herbivore removal. Additionally, the generalist slug and the generalist caterpillar preferred and disliked the same plant species, indicating that they perceive the balance between defense and nutritional value similarly. We conclude that the growth-defense trade-off in grassland species acts via the preference of herbivores and that among-species variation in plant growth and preference to herbivores drives plant community composition.