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Plants produce a great number of phytochemical compounds mediating a variety of different functions. Recently, phytochemical diversity (chemodiversity), a way which to quantify the complex phenotype formed by sets of phytochemicals, has been suggested to be important for function. However, no study has systematically examined the potential (in)direct functional importance of chemodiversity on a general level, partly due to a lack of an agreement on how to quantify this aspect of the plant phenotype. This paper has four aims: 1) We discuss how chemodiversity (deconstructed into components of richness, evenness and disparity) may quantify different aspects of the phenotype that are ecologically relevant. 2) We systematically review the literature on chemodiversity to examine methodological practices, explore ecological patterns of variability in diversity across different levels of biological organization, and investigate the functional role of this diversity in interactions between plants and other organisms. 3) We provide a framework facilitating decisions on which measure of chemodiversity is best used in different contexts. 4) We outline open questions and avenues for future research in this area. A more thorough understanding of phytochemical diversity will increase our knowledge on the functional role phytochemical compounds, and how they shape ecological interactions between plants and their environment.

IntroductionAssessing 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.ObjectivesIn 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 usedTanacetum 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.MethodsMonoclonal 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.ResultsChemotype-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.ConclusionOur 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.

This review comprehensively summaries the structure diversity and possible biosynthesis of representative Ganoderma meroterpenoids (GMs), as well as the chemical synthesis and pharmacological activities of some significant GMs.

14 páginas, 3 figuras, 2 tablas, 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, We thank Nicole M. van Dam, Henriette Uthe, Fredd Vergara, Martin Volf, and Alexander Weinhold for their valuable advice on metabolite analyses as well as Beate Rothe and Michael Reichelt for their help with chemical analyses. Moreover, comments by two anonymous reviewers helped to improve the paper. This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 677232). Further support came from the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig and the EcoMetEoR platform, funded by the German Research Foundation (FZT 118). AMM acknowledges support from the program for attracting talent to Salamanca from Fundación Salamanca Ciudad de Cultura y Saberes and Ayuntamiento de Salamanca.

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)-β-ocimene and (E)-β-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)-β-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.

Terpene synthases (TPSs) and trans-isoprenyl diphosphate synthases (IDSs) are among the core enzymes for creating the enormous diversity of terpenoids. Despite having no sequence homology, TPSs and IDSs share a conserved “α terpenoid synthase fold” and a trinuclear metal cluster for catalysis, implying a common ancestry with TPSs hypothesized to evolve from IDSs anciently. Here we report on the identification and functional characterization of novel IDS-like TPSs (ILTPSs) in fungi that evolved from IDS relatively recently, indicating recurrent evolution of TPSs from IDSs. Through large-scale bioinformatic analyses of fungal IDSs, putative ILTPSs that belong to the geranylgeranyl diphosphate synthase (GGDPS) family of IDSs were identified in three species of Melampsora. Among the GGDPS family of the two Melampsora species experimentally characterized, one enzyme was verified to be bona fide GGDPS and all others were demonstrated to function as TPSs. Melampsora ILTPSs displayed kinetic parameters similar to those of classic TPSs. Key residues underlying the determination of GGDPS versus ILTPS activity and functional divergence of ILTPSs were identified. Phylogenetic analysis implies a recent origination of these ILTPSs from a GGDPS progenitor in fungi, after the split of Melampsora from other genera within the class of Pucciniomycetes. For the poplar leaf rust fungus Melampsora larici-populina, the transcripts of its ILTPS genes were detected in infected poplar leaves, suggesting possible involvement of these recently evolved ILTPS genes in the infection process. This study reveals the recurrent evolution of TPSs from IDSs since their ancient occurrence and points to the possibility of a wide distribution of ILTPS genes in three domains of life.

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.

An extraordinarily high intraspecific chemical diversity, i.e. chemodiversity, has been found in several plant species, of which some are of major ecological or economic relevance. Moreover, even within an individual plant there is substantial chemodiversity among tissues and across seasons. This chemodiversity likely has pronounced ecological effects on plant mutualists and antagonists, associated foodwebs and, ultimately, biodiversity. Surprisingly, studies on interactions between plants and their herbivores or pollinators often neglect plant chemistry as a level of diversity and phenotypic variation. The main aim of this Research Unit (RU) is to understand the emergence and maintenance of intraspecific chemodiversity in plants. We address the following central questions: 1) How does plant chemodiversity vary across levels, i.e., within individuals, among individuals within populations, and among populations? 2) What are the ecological consequences of intraspecific plant chemodiversity? 3) How is plant chemodiversity genetically determined and maintained? By combining field and laboratory studies with metabolomics, transcriptomics, genetic tools, statistical data analysis and modelling, we aim to understand causes and consequences of plant chemodiversity and elucidate its impacts on the interactions of plants with their biotic environment. Furthermore, we want to identify general principles, which hold across different species, and develop meaningful measures to describe the fascinating diversity of defence chemicals in plants. These tasks require integrated scientific collaboration of experts in experimental and theoretical ecology, including chemical and molecular ecology, (bio)chemistry and evolution.

Plants have evolved a vast variety of secondary metabolites to counteract biotic stress. Volatile organic compounds (VOCs) are carbon-based molecules induced by herbivore attack or pathogen infection. A mixture of plant VOCs is released for direct or indirect plant defense, plant-plant or plant-insect communication. Recent studies suggest that VOCs can also induce biotic stress resistance in distant organs and neighboring plants. Among other VOCs, green leaf volatiles (GLVs) are quickly released by plant tissue after the onset of herbivory or wounding.We analysed VOCs emitted by 13-day old barley plants (Hordeum vulgare L.) after mechanical wounding using passive absorbers and TD-GC/MS detection. We investigated the influence of pure (Z)-3-hexenyl acetate (Z3HAC) as well as complex VOCs from wounded barley plants on the barley - powdery mildew interaction by pre-exposure in a static and a dynamic headspace connected to a powdery mildew susceptibility assay.GLVs dominated the volatile profile of wounded barley plants with Z3HAC as the most prominent compound. Pre-exposure with Z3HAC resulted in induced resistance of barley against fungal infection. Barley complex volatiles emitted after mechanical wounding, similarly, enhanced resistance in receiver plants.We found volatile-induced modification of the interaction towards an enhanced resistance against fungal infection. In addition, Z3HAC triggered a modulation of the alcohol dehydrogenase isoenzyme activity in receiver plants, a physiological response that possibly contributes to induced resistance. Plant-originated volatile metabolites could be a useful supplementation for future agronomic or horticultural practices.HighlightVolatile-induced modification of the barley-powdery mildew interaction towards an enhanced resistance against fungal infection.

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.

Secondary metabolites play important roles in plant responses to environmental stress but may also represent a large carbon (C) cost, resulting in trade-offs with other C sinks like growth and storage. However, it remains uncertain how such trade-offs may vary with changes in resource availability including water and CO2 availability. We conducted a glasshouse experiment with peppermint (Mentha x piperita L.) exposed to four treatments: control (sufficient irrigation and near-ambient CO2), water deficit (50% irrigation and near-ambient CO2), CO2 limitation (sufficient irrigation and below-ambient CO2) and the combination of water and CO2 deficits. Continuous 13CO2 labelling was used to trace allocation of newly-assimilated C. Concentrations of soluble sugars significantly increased under water deficit but decreased along with aboveground biomass under low CO2, while monoterpene concentrations remained relatively constant, independent of treatments. Under water deficit, there were no differences in allocation of new vs old C to monoterpenes production, structural growth and storage; plants grown under low CO2 even invested proportionally more newly-assimilated C for monoterpenes production, suggesting a preferential allocation to defense at the expense of growth. We concluded that C allocation to monoterpenes is actively regulated in coordination with growth and storage under water and C stresses, consistent with an optimal defense strategy to protect young tissues.

Trees are large organisms that structure forest ecosystems by providing an environment for an enormous diversity of animal, microbial and plant species. As these species use trees as their common hosts, many are likely to interact with each other directly or indirectly. From studies on herbaceous plant species we know that microbes can affect the interaction of plants with herbivorous insects, for example via changes in plant metabolite profiles. The consequences of fungal colonization for tree-insect interactions are, however, barely known, despite the importance of these ecological communities. In this review we explore the interaction of leaf-inhabiting pathogenic and endophytic fungi with trees and the consequences for tree-living insect herbivores. We discuss molecular, physiological, chemical, biochemical and ecological aspects of tree-fungus interactions and summarize the current knowledge on the direct and indirect effects of tree-inhabiting fungi on insect herbivores. Our mechanistic understanding of the tripartite interaction of trees with leaf-inhabiting fungi and insect herbivores is still in its infancy. We are currently facing substantial drawbacks in experimental methodology that prevent us from revealing the effect of one single fungal species on a particular insect herbivore species and vice versa. Future studies applying a versatile toolbox of modern molecular, chemical analytical and ecological techniques in combined laboratory and field experiments will unequivocally lead to a better understanding of fungus-tree-insect interactions.

Recent research shows that earthworms can alter defense traits of plants against herbivores and pathogens by affecting soil biochemistry. Yet, the effects of invasive earthworms on defense traits of native plants from previously earthworm‐free ecosystems as well as the consequences for multitrophic interactions are virtually unknown.Here we use a combination of an observational study and a complementary experimental study to investigate the effects of invasive earthworms on leaf defense traits, herbivore damage and pathogen infection in two poplar tree species (Populus balsamifera and Populus tremuloides) native to North American boreal forests.Our observational study showed that earthworm invasion was associated with enhanced leaf herbivory (by leaf‐chewing insects) in saplings of both tree species. However, we only detected significant shifts in the concentration of chemical defense compounds in response to earthworm invasion for P. balsamifera. Specifically, leaf phenolic concentrations, including salicinoids and catechin, were lower in P. balsamifera from earthworm‐invaded sites.Our experimental study confirmed an earthworm‐induced reduction in leaf defense levels in P. balsamifera for one of the defense compounds, tremulacin. The experimental study additionally showed that invasive earthworms reduced leaf dry matter content, potentially increasing leaf palatability, and enhanced susceptibility of trees to infection by a fungal pathogen, but not to aphid infestation, in the same tree species. Synthesis. Our results show that invasive earthworms can decrease the concentrations of some chemical defense compounds in P. balsamifera, which could make them susceptible to leaf‐chewing insects. Such potential impacts of invasive earthworms are likely to have implications for tree survival and competition, native tree biodiversity and ecosystem functioning., Invasive earthworms can dramatically alter native biodiversity and ecosystem functioning. Yet, we know little how invasive earthworms affect the chemical defense of native trees. Here using a combination of observational and experimental studies, we show that invasive earthworms from Europe can decrease the concentrations of some chemical defense compounds in a native North American deciduous tree, which could make them susceptible to leaf‐chewing insects.

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.

Flavan-3-ols including the monomeric catechin and the polymeric proanthocyanidins (PAs) are abundant phenolic metabolites in poplar (Populus spp.) previously described to protect leaves against pathogen infection. However, it is not known whether stems are also defended in this way. Here we investigated flavan-3-ol accumulation, activity, and the regulation of formation in black poplar (P. nigra) stems after infection by a newly described fungal stem pathogen, Plectosphaerella populi, which forms canker-like lesions in stems. We showed that flavan-3-ol contents increased in P. populi-infected black poplar stems over the course of infection compared to non-infected controls. Transcripts of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes involved in the last steps of flavan-3-ol biosynthesis were also upregulated upon fungal infection indicating de novo biosynthesis. Amending culture medium with catechin and PAs reduced the mycelial growth of P. populi, suggesting that these metabolites act as anti-pathogen defenses in poplar in vivo. Among the hormones, salicylic acid (SA) was higher in P. populi-infected tissues compared to the non-infected controls over the course of infection studied, while jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels were higher than controls only at the early stages of infection. Interestingly, cytokinins (CKs) were also upregulated in P. populi-infected stems. Poplar saplings treated with CK showed decreased levels of flavan-3-ols and SA in stems suggesting a negative association between CK and flavan-3-ol accumulation. Taken together, the sustained upregulation of SA in correlation with catechin and PA accumulation suggests that this is the dominant hormone inducing the formation of antifungal flavan-3-ols during P. populi infection of poplar stems.

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.

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.

Volatile organic compounds (VOCs) released from plants are known to mediate indirect defense against herbivores and trigger intra- and interplant signaling. While systemic defense response can be mediated both via volatile and vascular signals, it is not clear whether common ancestry and/or plant growth forms influence the choice of either mode in planta. We hypothesize that larger woody plants with a complex anatomy should rely more on volatile-mediated signaling, apparently to circumvent vascular restrictions that slow down the communication over a large distance. On the other hand, in smaller herbaceous plants faster systemic response can be achieved via vascular signaling. To investigate whether plant VOCs emission is related to plant phylogeny or growth form, we studied the composition of herbivory-induced plant volatiles in 13 Brassicaceae species representing all four evolutionary lineages, because this family is characterized by both a well-resolved phylogeny and highly diverse growth forms. Our results revealed that woody species consistently emitted a more complex blend of volatiles than herbaceous species. However, phylogenetic relatedness of the species did not explain the observed volatile emission patterns. This emphasizes the influence of growth form, rather than phylogenetic relationships on the variation in plant volatile emissions. Our findings suggest that woody, perennial plant species emit diverse VOCs, likely because these compounds comprise a more efficient mode of defense response in these large, anatomically complex plants.

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., The project was supported by the Graduate School ‘Evolutionary Networks: Organisms, Reactions, Molecules’ (E‐Norm) of the Heinrich‐Heine‐University, Düsseldorf, Germany, the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG JU 2856/1‐1 and DFG JU 2856/2‐2).

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.

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., Plants communicate with other organisms through volatile organic compounds. Deciphering this language is important for our knowledge on plant communication. We show that the ability of a compound to serve as cue for insect herbivory is affected by its physicochemical and physiological properties.

Volatiles are often released upon herbivory as plant defense compounds. While the formation of volatiles above-ground has been intensively studied, little is known about herbivore-induced root volatiles. Here, we show that cockchafer larvae-damaged roots of Populus trichocarpa and P. nigra release a mixture of monoterpenes, including (−)-α-pinene, (−)-camphene, (−)-β-pinene, p-cymene, and 1,8-cineole. Three terpene synthases, PtTPS16 and PtTPS21 from P. trichocarpa and PnTPS4 from P. nigra, could be identified and characterized in vitro. PnTPS4 was found to produce 1,8-cineole as sole product. PtTPS16 and PtTPS21, although highly similar to each other, showed different product specificities and produced γ-terpinene and a mixture of (−)-camphene, (−)-α-pinene, (−)-β-pinene, and (−)-limonene, respectively. Four active site residues were found to determine the different product specificities of the two enzymes. The expression profiles of PtTPS16, PtTPS21, and PnTPS4 in undamaged and herbivore-damaged poplar roots generally matched the emission pattern of monoterpenes, indicating that monoterpene emission in roots is mainly determined at the gene transcript level. Bioassays with Phytophtora cactorum (Oomycetes) revealed inhibitory effects of vapor-phase 1,8-cineole and (−)-β-pinene on the growth of this important plant pathogen. Thus herbivore-induced volatile monoterpenes may have a role in defense against pathogens that cause secondary infections after root wounding.

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. Electronic supplementary material The online version of this article (10.1186/s12870-018-1541-9) contains supplementary material, which is available to authorized users.

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.

Herbivory is well known to trigger increased emission of volatile organic compounds (VOCs) from plants, but we know little about the responses of mature trees. We measured the volatiles emitted by leaves of old-growth black poplar (Populus nigra) trees after experimental damage by gypsy moth (Lymantria dispar) caterpillars in a floodplain forest, and studied the effect of herbivory on the transcript abundance of two genes involved in the biosynthesis of VOCs, and the accumulation of defence phytohormones. Herbivory significantly increased volatile emission from the experimentally damaged foliage, but not from adjacent undamaged leaves in the damaged branches (i.e., no systemic response). Methylbutyraldoximes, 4,8-dimethyl-1,3,7-nonatriene (DMNT), (Z)-3-hexenol and (E)-β-ocimene, amongst other compounds, were found to be important in distinguishing the blend of herbivore-damaged vs. undamaged leaves. Herbivory also increased expression of PnTPS3 (described here for the first time) and PnCYP79D6-v4 genes at the damaged sites, these genes encode for an (E)-β-ocimene synthase and a P450 enzyme involved in aldoxime formation, respectively, demonstrating de novo biosynthesis of the volatiles produced. Herbivore-damaged leaves had significantly higher levels of jasmonic acid and its conjugate (−)-jasmonic acid-isoleucine. This study shows that mature trees in the field have a robust response to herbivory, producing induced volatiles at the damaged sites even after previous natural herbivory and under changing environmental conditions, however, further studies are needed to establish whether the observed absence of systemic responses is typical of mature poplar trees or if specific conditions are required for their induction.

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. Electronic supplementary material The online version of this article (doi:10.1007/s10886-016-0698-7) contains supplementary material, which is available to authorized users.

As dead wood is ephemeral, saproxylic insect species must be able to effectively locate new resources via, e.g. olfactory cues. 2. Although saproxylic flat bugs feed on fungal mycelia, published evidence points to their use of dead wood-specific volatile cues rather than fungal-specific cues for long-distance orientation towards new habitat patches. 3. To evaluate which olfactory cues saproxylic flat bugs use for short-distance orientation, we tested the attraction of Aradus obtectus (Vasarhelyi) to fresh wood, fungal mycelia and fungal fruiting bodies using an olfactometer. 4. Aradus obtectus individuals were attracted only to fungal mycelia but not to wood or fungal fruiting bodies. 5. Our results and those of our earlier field experiment testing long-distance attraction (Seibold et al., 2014) together suggest that A. obtectus uses cues emit- ted by dead wood to locate potential resources over long distances, but relies on olfactory cues emitted by mycelia of its host species to locate suitable short- range microhabitats.

Chemoecology, 22 (2), ISSN:0937-7409, ISSN:1423-0445

Novel species of fungi described in the present study include the following from Malaysia: Castanediella eucalypti from Eucalyptus pellita, Codinaea acacia from Acacia mangium, Emarcea eucalyptigena from Eucalyptus brassiana, Myrtapenidiella eucalyptorum from Eucalyptus pellita, Pilidiella eucalyptigena from Eucalyptus brassiana and Strelitziana malaysiana from Acacia mangium. Furthermore, Stachybotrys sansevieriicola is described from Sansevieria ehrenbergii (Tanzania), Phacidium grevilleae from Grevillea robusta (Uganda), Graphium jumulu from Adansonia gregorii and Ophiostoma eucalyptigena from Eucalyptus marginata (Australia), Pleurophoma ossicola from bone and Plectosphaerella populi from Populus nigra (Germany), Colletotrichum neosansevieriae from Sansevieria trifasciata, Elsinoë othonnae from Othonna quinquedentata and Zeloasperisporium cliviae (Zeloasperisporiaceae fam. nov.) from Clivia sp. (South Africa), Neodevriesia pakbiae, Phaeophleospora hymenocallidis and Phaeophleospora hymenocallidicola on leaves of a fern (Thailand), Melanconium elaeidicola from Elaeis guineensis (Indonesia), Hormonema viticola from Vitis vinifera (Canary Islands), Chlorophyllum pseudoglobossum from a grassland (India), Triadelphia disseminata from an immunocompromised patient (Saudi Arabia), Colletotrichum abscissum from Citrus (Brazil), Polyschema sclerotigenum and Phialemonium limoniforme from human patients (USA), Cadophora vitícola from Vitis vinifera (Spain), Entoloma flavovelutinum and Bolbitius aurantiorugosus from soil (Vietnam), Rhizopogon granuloflavus from soil (Cape Verde Islands), Tulasnella eremophila from Euphorbia officinarum subsp. echinus (Morocco), Verrucostoma martinicensis from Danaea elliptica (French West Indies), Metschnikowia colchici from Colchicum autumnale (Bulgaria), Thelebolus microcarpus from soil (Argentina) and Ceratocystis adelpha from Theobroma cacao (Ecuador). Myrmecridium iridis (Myrmecridiales ord. nov., Myrmecridiaceae fam. nov.) is also described from Iris sp. (The Netherlands). Novel genera include (Ascomycetes): Budhanggurabania from Cynodon dactylon (Australia), Soloacrosporiella, Xenocamarosporium, Neostrelitziana and Castanediella from Acacia mangium and Sabahriopsis from Eucalyptus brassiana (Malaysia), Readerielliopsis from basidiomata of Fuscoporia wahlbergii (French Guyana), Neoplatysporoides from Aloe ferox (Tanzania), Wojnowiciella, Chrysofolia and Neoeriomycopsis from Eucalyptus (Colombia), Neophaeomoniella from Eucalyptus globulus (USA), Pseudophaeomoniella from Olea europaea (Italy), Paraphaeomoniella from Encephalartos altensteinii, Aequabiliella, Celerioriella and Minutiella from Prunus (South Africa). Tephrocybella (Basidiomycetes) represents a novel genus from wood (Italy). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa., Christian Lechat gratefully acknowledges Cesar Delnatte (DEAL, Martinique) fro the identification of the horst of Verrucostoma martinicensis. Alfredo Vizzini thanks Edmondo Grilli (Pescara, Italy), for comments on the text of Tephrocybella griseonigrescens. Dilnora Gouliamova was supported by a grant (D002-TK-176) from the Bulgarian Science Fund, EU F6 Synthesis program. She is grateful to K. Metodiev for permission to use the picture of Colchicum autumnale (http://www.bgflora. net/). Olga V. Morozova, Eugene S. Popov and Xiao-Lan He are grateful to V. Trunov, T.H. Li, C.Y. Deng, H. Huang and Y.W. Xia for valuable Entoloma collections. Alina V. Alexandrova was supported by the Russian Science Foundation (project N 14-50-00029). Ekaterina F. Malysheva, Olga V. Morozova, Alexander E. Kovalenko and Eugene S. Popov acknowledge financial support from the Russian Foundation for Basic Research (project 13-04-00838a and 15-04-04645a). Margarita Dueñas, María P. Martín and M. Teresa Telleria acknowledge financial support from the Plan Nacional I+D+I projects No. CGL2009-07231 and CGL2012-3559. They are also thankful to Marian Gleen (Seton Hall University, USA) for commenting on the text. Cony Decock gratefully acknowledges the financial support received from the FNRS / FRFC (convention FRFC 2.4544.10), the CNRS-French Guiana and the Nouragues staff, which enabled fieldwork in French Guiana, and the Belgian State – Belgian Federal Science Policy through the BCCMTM research programme. We also thank the CBS technical staff, A. van Iperen (cultures), M. Vermaas (photographic plates) and M. Starink-Willemse (DNA isolation, amplification and sequencing) for their invaluable assistance.

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.

Plants emit complex mixtures of volatile organic compounds from floral and vegetative tissue, especially after herbivore damage, so it is difficult to associate individual compounds with activity towards pollinators, herbivores or herbivore enemies. Attention has usually focused upon the biological activity of the most abundant compounds; but here, we detail a number of reports implicating minor volatiles in attractant or deterrent roles. This is not surprising given the exquisite sensitivity of insect olfactory systems for certain substances. In this context, it is worth reconsidering the methods involved in sampling volatile compounds from plants, measuring their abundance and determining their biological activity to ensure that minor compounds are not overlooked. Here, we describe various experimental approaches and chemical and statistical methods that should increase the chance of detecting minor compounds with major biological activities.

After herbivory, plants release volatile organic compounds from damaged foliage as well as from nearby undamaged leaves that attract herbivore enemies. Little is known about what controls the volatile emission differences between damaged and undamaged tissues and how these affect the orientation of herbivore enemies. We investigated volatile emission from damaged and adjacent undamaged foliage of black poplar (Populus nigra) after herbivory by gypsy moth (Lymantria dispar) caterpillars and determined the compounds mediating the attraction of the gypsy moth parasitoid Glyptapanteles liparidis (Braconidae). Female parasitoids were more attracted to gypsy moth-damaged leaves than to adjacent non-damaged leaves. The most characteristic volatiles of damaged versus neighbouring undamaged leaves included terpenes, green leaf volatiles and nitrogen-containing compounds, such as aldoximes and nitriles. Electrophysiological recordings and olfactometer bioassays demonstrated the importance of nitrogenous volatiles. Under field conditions, parasitic Hymenoptera were more attracted to traps baited with these substances than most other compounds. The differences in volatile emission profiles between damaged and undamaged foliage appear to be regulated by jasmonate signalling and the local activation of volatile biosynthesis. We conclude that characteristic volatiles from damaged black poplar foliage are essential cues enabling parasitoids to find their hosts.

Transgenic hybrid aspen (Populus tremula x tremuloides) overexpressing the MYB134 tannin regulatory gene show dramatically enhanced condensed tannin (proanthocyanidin) levels, as well as shifts in other phenolic metabolites. A series of insect bioassays with forest tent caterpillars (Malacosoma disstria) and gypsy moth (Lymantria dispar) caterpillars was carried out to determine how this metabolic shift affects food preference and performance of generalist tree-feeding lepidopterans. Both species showed a distinct preference for the high-tannin MYB134 overexpressor plants, and L. dispar performance was enhanced relative to controls. L. dispar reached greater pupal weight and showed reduced time to pupation when reared on the MYB134 overexpressing poplar. These results were unexpected since enhanced condensed tannin levels were predicted to act as feeding deterrents. However, the data may be explained by the observed decrease in the salicinoids (phenolic glycosides) salicortin and tremulacin that accompanied the upregulation of the condensed tannins in the transgenics. We conclude that for these two lepidopteran species, condensed tannin levels are unlikely to be a major determinant of caterpillar food preference or performance. However, our experiments show that overexpression of a single regulatory gene in transgenic aspen can have a significant impact on herbivorous insects.

Species of the Salicaceae produce phenolic compounds that may function as anti-herbivore defenses. Levels of these compounds have been reported to increase upon herbivory, but only rarely have these changes in phenolics been studied under natural conditions. We profiled the phenolics of old-growth black poplar (Populus nigra L.) and studied the response to gypsy moth (Lymantria dispar L.) herbivory in two separate field experiments. In a first experiment, foliar phenolics of 20 trees were monitored over 4 weeks after caterpillar infestation, and in a second experiment the bark and foliar phenolics of a single tree were measured over a week. Of the major groups of phenolics, salicinoids (phenolic glycosides) showed no short term response to caterpillar feeding, but after 4 weeks they declined up to 40 % in herbivore damaged and adjacent undamaged leaves on the same branch when compared to leaves of control branches. Flavonol glycosides, low molecular weight flavan-3-ols, and condensed tannins were not affected by herbivory in the first experiment. However, in the single-tree experiment, foliar condensed tannins increased by 10-20 % after herbivory, and low molecular weight flavan-3-ols decreased by 10 % in the leaves but increased by 10 % in the bark. Despite 15 % experimental leaf area loss followed by a 5-fold increase in foliar jasmonate defense hormones, we found no evidence for substantial induction of phenolic defense compounds in old growth black poplar trees growing in a native stand. Thus, if phenolics in these trees function as defenses against herbivory, our results suggest that they act mainly as constitutive defenses.

The survival of insect herbivores on chemically defended plants may often depend on their ability to metabolize these defense compounds. However, only little knowledge is available on how insects actually process most plant defense compounds. We investigated the metabolism of salicinoids, a major group of phenolic glycosides in poplar and willow species, by a generalist herbivore, the gypsy moth (Lymantria dispar). Seven salicinoid metabolites identified in gypsy moth caterpillar feces were mostly conjugates with glucose, cysteine or glycine. Two of the glucosides were phosphorylated, a feature not previously reported for insect metabolites of plant defense compounds. The origins of these metabolites were traced to specific moieties of three major poplar salicinoids ingested, salicin, salicortin and tremulacin. Based on the observed metabolite patterns we were able to deduce the initial steps of salicinoid breakdown in L. dispar guts, which involves cleavage of ester bonds. The conjugated molecules were effectively eliminated within 24 h after ingestion. Some of the initial breakdown products (salicin and catechol) demonstrated negative effects on insect growth and survival in bioassays on artificial diets. Gypsy moth caterpillars with prior feeding experience on salicinoid-containing poplar foliage converted salicinoids to the identified metabolites more efficiently than caterpillars pre-fed an artificial diet. The majority of the metabolites we identified were also produced by other common poplar-feeding insects. The conversion of plant defenses like salicinoids to a variety of water-soluble sugar, phosphate and amino acid conjugates and their subsequent excretion fits the general detoxification strategy found in insect herbivores and other animals.

This study has been supported by the ANR Grant No. ANR-05-JCJC-0203-01 and the Region Centre Project 201000047141; International audience; Phytohormones have long been hypothesized to play a key role in the interactions betweenplant-manipulating organisms and their host–plants such as insect–plant interactions that lead to gallor ‘green-islands’ induction. However, mechanistic understanding of how phytohormones operate inthese plant reconfigurations is lacking due to limited information on the molecular and biochemical phytohormonalmodulation following attack by plant-manipulating insects. In an attempt to fill this gap, thepresent study provides an extensive characterization of how the leaf-miner Phyllonorycter blancardellamodulates the major phytohormones and the transcriptional activity of plant cells in leaves of Malusdomestica.We show here, that cytokinins strongly accumulate in mined tissues despite a weak expressionof plant cytokinin-related genes. Leaf-mining is also associated with enhanced biosynthesis of jasmonicacid precursors but not the active form, a weak alteration of the salicylic acid pathway and a clear inhibitionof the abscisic acid pathway. Our study consolidates previous results suggesting that insects mayproduce and deliver cytokinins to the plant as a strategy to manipulate the physiology of the leaf to createa favorable nutritional environment. We also demonstrate that leaf-mining by P. blancardella leads to astrong reprogramming of the plant phytohormonal balance associated with increased nutrient mobilization,inhibition of leaf senescence and mitigation of plant direct and indirect defense

A sample of 1834 leaves from 83 plants in 26 families was collected in tropical rain forest in Madagascar from three vertical strata: top of emergent trees (up to 37 m), top of trees in upper canopy (about 22 m), and shrubs and saplings at ground level. These leaves were examined for damage by seven different agents: fungi, epiphyllae, mechanical injury, galls, leaf miners, grazing insects and skeletonizing insects. Fungi affected more than 60% of the leaves and grazing insects 45–65%, with other agents each affecting 3–30%; fewer than 4% of the leaves escaped unscathed by any agent. Individual leaves were attacked by up to five agents. There was a sharp decline in proportion of leaves affected by fungi, leaf miners, epiphyllae and mechanical breakage with increasing category of severity. Grazing insects, mechanical injury, and perhaps galls, had greater impact at ground level than in the canopy, with fungi and skeletonizing insects showing the opposite pattern. Leaf miners had lower incidence in the canopy than elsewhere. The observed vertical stratification means that a tree not only needs to balance its defences to meet multiple threats in any stratum, but must adapt to a different suite of challenges during its lifetime. Attack by grazing insects and fungi are major challenges for saplings at ground level, but with increasing height above the ground fungal attack on leaves becomes more prevalent, but attack by grazing insects less so.

Herbivore damage to leaves and other vegetative tissues often stimulates the emission of volatile compounds, suggesting that these substances have a role in plant defense. In fact, ample evidence has accumulated in the last few years indicating that volatiles from vegetative plant parts can directly repel herbivores, such as ovipositing butterflies and host-seeking aphids. Volatiles have also been demonstrated to protect plants by attracting herbivore enemies, such as parasitic wasps, predatory arthropods and possibly even insectivorous birds. Even below ground herbivory results in the release of volatiles that attract herbivore enemies. However, plant volatiles are also known to attract enemies of plants. Hence, to determine the true value of these substances in defense, more research is needed especially in natural communities with non-agricultural species.

1. Polymorphism has been described for a number of herbivorous insects, but little is known about whether differences in body colour cause fitness differences. In Chorthippus parallelus, three main colour morphs occur, namely brown, green, and dorsally striped. 2. The present study examined colour morph abundances and morph-related differences in body size, oviposition rate, and offspring numbers in females of C. parallelus collected in 15 montane grasslands. The study also examined the effect of plant species richness, composition, community productivity, and solar radiation on colour morph frequency and fitness. 3. The relative frequencies of the three colour morphs was 31.7% (brown), 33.1% (green), and 35.2% (dorsally striped), but the morphs were not evenly distributed across the 15 sites. 4. There was no effect of the habitat variables on the distribution of the green and the striped morph in the study sites, however 80% of the variation in the abundances of the brown morph was explained by plant species richness and composition. 5. Grasshopper size was equal among the morphs. Brown females laid significantly more egg pods than the green and dorsally striped morphs. There were no significant differences in offspring numbers among the colour morphs. 6. Body colour in C. parallelus seems to be a fitness-relevant trait, raising the question of the evolutionary maintenance of polymorphism.

Generalist insect herbivores may profit by feeding on a mixture of plant species that differ in nutritional quality. Herbivore performance can also be affected by intraspecific host plant variation. However, it is unknown whether conspecific plant individuals differ sufficiently to promote diet-mixing behavior in specialist herbivores. We experimentally tested this "specialist diet-mixing hypothesis" for specialist caterpillars (Chrysopsyche imparilis, Lasiocampidae) in a West African savanna. The caterpillars switched regularly between host tree individuals (Combretum fragrans, Combretaceae). To examine whether switching benefited caterpillar performance via diet-mixing, the caterpillars were reared either on leaves from several plant individuals (mixed diet) or on leaves from a single plant. The strongest effect of diet-mixing was found for fecundity, with females reared on a mixed diet laying significantly more eggs than sisters receiving a single-plant diet. In addition, a mixed diet decreased variability in egg size and increased the growth of second-instar caterpillars. Supplementary food choice experiments were conducted to assess a potential influence of lowered host quality (induced by herbivory) on caterpillar behavior; no such effect was found. By linking intraspecific host-switching behavior and herbivore performance, this study provides new information on the relevance of intraspecific plant variation for herbivorous insects.

Herbivore-induced plant volatile emission is often considered to be attacker species-specific, but most experimental evidence comes from short lived herbaceous species. In a recent study we showed that black poplar (Populus nigra) trees emit a complex blend of volatiles from damaged leaves when they are attacked by generalist gypsy moth (Lymantria dispar) caterpillars. Minor nitrogenous volatiles were especially characteristic of this blend. Here we show that attack on P. nigra by a beetle species, Phratora vulgatissima (Coleoptera, Chrysomelidae), led to the emission of the same compounds as already observed after caterpillar herbivory, but with striking quantitative changes in the blend. The consequences for attraction of herbivore enemies are discussed.