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.

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.

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.

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.

Invasive earthworms reduce chemical defense and increase herbivory and pathogen infection in native trees.

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.

Evolution of isoprenyl diphosphate synthase-like terpene synthases in fungi.

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.

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.

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.

Accumulation of Catechin and Proanthocyanidins in Black Poplar Stems After Infection by Plectosphaerella populi: Hormonal Regulation, Biosynthesis and Antifungal Activity.

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.

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.

Herbivore-induced volatile emission from old-growth black poplar trees under field conditions.

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)-ß-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)-ß-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.

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.

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.

The occurrence and formation of monoterpenes in herbivore-damaged poplar roots.

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, (-)-ß-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, (-)-ß-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 (-)-ß-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.

Rust Infection of Black Poplar Trees Reduces Photosynthesis but Does Not Affect Isoprene Biosynthesis or Emission.

Poplar (Populus spp.) trees are widely distributed and play an important role in ecological communities and in forestry. Moreover, by releasing high amounts of isoprene, these trees impact global atmospheric chemistry. One of the most devastating diseases for poplar is leaf rust, caused by fungi of the genus Melampsora. Despite the wide distribution of these biotrophic pathogens, very little is known about their effects on isoprene biosynthesis and emission. We therefore infected black poplar (P. nigra) trees with the rust fungus M. larici-populina and monitored isoprene emission and other physiological parameters over the course of infection to determine the underlying mechanisms. We found an immediate and persistent decrease in photosynthesis during infection, presumably caused by decreased stomatal conductance mediated by increased ABA levels. At the same time, isoprene emission remained stable during the time course of infection, consistent with the stability of its biosynthesis. There was no detectable change in the levels of intermediates or gene transcripts of the methylerythritol 4-phosphate (MEP) pathway in infected compared to control leaves. Rust infection thus does not affect isoprene emission, but may still influence the atmosphere via decreased fixation of CO2.

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.

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.