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Plants produce many secondary metabolites showing considerable inter- and intraspecific diversity of concentration and composition as a strategy to cope with environmental stresses. The evolution of plant defenses against herbivores and pathogens can be unraveled by understanding the mechanisms underlying chemical diversity. Pyrrolizidine alkaloids are a class of secondary metabolites with high diversity. We performed a qualitative and quantitative analysis of 80 pyrrolizidine alkaloids with liquid chromatography-tandem mass spectrometry of leaves from 17 Jacobaea species including one to three populations per species with 4–10 individuals per population grown under controlled conditions in a climate chamber. We observed large inter- and intraspecific variation in pyrrolizidine alkaloid concentration and composition, which were both species-specific. Furthermore, we sequenced 11 plastid and three nuclear regions to reconstruct the phylogeny of the 17 Jacobaea species. Ancestral state reconstruction at the species level showed mainly random distributions of individual pyrrolizidine alkaloids. We found little evidence for phylogenetic signals, as nine out of 80 pyrrolizidine alkaloids showed a significant phylogenetic signal for Pagel's λ statistics only, whereas no significance was detected for Blomberg's K measure. We speculate that this high pyrrolizidine alkaloid diversity is the result of the upregulation and downregulation of specific pyrrolizidine alkaloids depending on ecological needs rather than gains and losses of particular pyrrolizidine alkaloid biosynthesis genes during evolution.

Based on the hypothesis that the variation of the metabolomes of latex is a response to selective pressure and should thus be affected differently from other organs, their variation could provide an insight into the defensive chemical selection of plants. Metabolic profiling was used to compare tissues of three Euphorbia species collected in diverse regions. The metabolic variation of latexes was much more limited than that of other organs. In all the species, the levels of polyisoprenes and terpenes were found to be much higher in latexes than in leaves and roots of the corresponding plants. Polyisoprenes were observed to physically delay the contact of pathogens with plant tissues and their growth. A secondary barrier composed of terpenes in latex and in particular, 24-methylenecycloartanol, exhibited antifungal activity. These results added to the well-known role of enzymes also present in latexes, show that these are part of a cooperative defense system comprising biochemical and physical elements. Supplementary Information The online version contains supplementary material available at 10.1007/s10886-021-01274-x.

AimsSoil biotic communities can strongly impact plant performance. So far, most studies on plant-soil-interactions have estimated the effect of the soil microbial community on plant mass after a fixed duration of plant growth. However, these interactions may change over time and several studies have argued that plant-soil interactions are more important for young seedlings than for older plants. In this paper we ask the question: how long-lasting the effect of the soil microbial community on plant growth is. This is important as the growth rate of a plant is not only determined by the growing conditions but also by the size of the plant itself. Therefore, plant with a reduced growth rate early in life, due to negative effects of the soil microbial community, may increase less in biomass for a much longer period even though the relative growth rates do not differ any longer.MethodsWe examined the plant growth rates at three stages: early growth (0-21 days), mid growth (22 to 42 days) and late growth (43 to 63 days). We performed two growth experiments with Jacobaea vulgaris lasting 49 and 63 days. Plants were grown in sterilized soil or in sterilized soil inoculated with natural dune soil. In a third experiment, we examined the effect of the timing of soil inoculation prior to planting on the (relative-) growth rate of J. vulgaris plants with four different timing treatments.Important findingsIn all experiments, differences in biomass of plants grown in sterilized soil and inoculated soil (live soil) increased throughout the experiment. Interestingly, linear regression models with ln transformed dry weight against time for younger plants and for older plants in sterilized soil and live soil, respectively, showed that the relative growth rate of plants in the sterilized soil was only significantly higher than that of plants in the live soil in the first two to three weeks. After that period there was no longer a negative effect of the live soil on the relative growth rate of plants. In the third experiment, plant biomass decreased with increasing time between inoculation and planting. Overall, our results show that plants of J. vulgaris grew less well in live soil than in sterilized soil. The negative effects of soil inoculation on plant mass appeared to extend over the whole growth period but arise from the negative effects on relative growth rates that occurred in the first weeks after planting when plants have only less than 5% of the mass they obtained after 42 days. Our study highlights the importance of examining relative growth rates rather than final biomass to estimate the effects of soil microbial communities on plants.

Abiotic and biotic properties of soil can influence growth and chemical composition of plants. Although it is well-known that soil microbial composition can vary greatly spatially, how this variation affects plant chemical composition is poorly understood. We grew genetically identical Jacobaea vulgaris in sterilized soil inoculated with live soil collected from four natural grasslands and in 100% sterilized soil. Within each grassland we sampled eight plots, totalling 32 different inocula. Two samples per plot were collected, leading to three levels of spatial variation: within plot, between and within grasslands. The leaf metabolome was analysed with 1H Nuclear magnetic resonance spectroscopy (NMR) to investigate if inoculation altered the metabolome of plants and how this varied between and within grasslands. Inoculation led to changes in metabolomics profiles of J. vulgaris in two out of four sites. Plants grown in sterilized and inoculated soils differed in concentrations of malic acid, tyrosine, trehalose and two pyrrolizidine alkaloids (PA). Metabolomes of plants grown in inoculated soils from different sites varied in glucose, malic acid, trehalose, tyrosine and in one PA. The metabolome of plants grown in soils with inocula from the same site was more similar than with inocula from distant sites. We show that soil influences leaf metabolomes. Performance of aboveground insects often depends on chemical composition of plants. Hence our results imply that soil microbial communities, via affecting aboveground plant metabolomes, can impact aboveground plant-insect food chains but that it is difficult to make general predictions due to spatial variation in soil microbiomes. Electronic supplementary material The online version of this article (10.1007/s10886-020-01156-8) contains supplementary material, which is available to authorized users.

BackgroundBulk segregant analysis (BSA) can help identify quantitative trait loci (QTLs), but this may result in substantial bycatch of functionally irrelevant genes.ResultsHere we develop a Gene Ontology-mediated approach to zoom in on specific genes located inside QTLs identified by BSA as implicated in a continuous trait. We apply this to a novel experimental system: flowering time in the giant woody Jersey kale, which we phenotyped in four bulks of flowering onset. Our inferred QTLs yielded tens of thousands of candidate genes. We reduced this by two orders of magnitude by focusing on genes annotated with terms contained within relevant subgraphs of the Gene Ontology. A pathway enrichment test then led to the circadian rhythm pathway. The genes that enriched this pathway are attested from previous research as regulating flowering time. Within that pathway, the genesCCA1,FT, andTSFwere identified as having functionally significant variation compared toArabidopsis. We validated and confirmed our ontology-mediated results through genome sequencing and homology-based SNP analysis. However, our ontology-mediated approach produced additional genes of putative importance, showing that the approach aids in exploration and discovery.ConclusionsOur method is potentially applicable to the study of other complex traits and we therefore make our workflows available as open-source code and a reusable Docker container.

The overall effect of a live soil inoculum collected from nature on plant biomass is often negative. One hypothesis to explain this phenomenon is that the overall net pathogenic effect of soil microbial communities reduces plant performance. Induced plant defenses triggered by the application of the plant hormones jasmonic acid (JA) and salicylic acid (SA) may help to mitigate this pathogenic effect of live soil. However, little is known about how such hormonal application to the plant affects the soil and how this, in turn, impacts plant growth. We grew four plant species in sterilized and inoculated live soil and exposed their leaves to two hormonal treatments (JA and SA). Two species (Jacobaea vulgaris and Cirsium vulgare) were negatively affected by soil inoculation. In these two species foliar application of SA increased biomass in live soil but not in sterilized soil. Two other species (Trifolium repens and Daucus carota) were not affected by soil inoculum and for these two species foliar application of SA reduced plant biomass in both the sterilized and live soil. Application of JA reduced plant biomass in both soils for all species. We subsequently carried out a multiple generation experiment for one of the plant species, J. vulgaris. In each generation, the live soil was a mixture of 10% soil from the previous generation and 90% sterilized soil and the same hormonal treatments were applied. The negative effects of live soil on plant biomass were similar in all four generations, and this negative effect was mitigated by the application of SA. Our research suggests that the application of SA can mitigate the negative effects of live soil on plant growth. Although the inoculum of soil containing a natural live soil microbial community had a strong negative effect on the growth of J. vulgaris, we found no evidence for an increase or decrease in negative plant-soil feedback in either the control or the SA treated plants. Also plant performance did not decrease consistently with succeeding generations.

Concentration of plant secondary metabolites (SMs) show seasonal variations. However, it is still not well understood how these abiotic and biotic factors influence the seasonal variations of SMs. In addition, it is of interest to know if and how SMs are reallocated to the different plant organs, in particular whether SMs are reallocated to the remaining tissues when biomass is lost, e.g., during winter. Here we used Jacobaea vulgaris, Jacobaea aquatica, two F1 and four F2 hybrids that differed in their pyrrolizidine alkaloids (PAs) bouquet as a study system. A series of clones of these genotypes were investigated during their vegetative stage spanning 14 months in a semi-natural environment. We found that the total PA concentration in roots and shoots showed a gradual increase until the spring of the second year, whereafter it dropped substantially in shoots. The variation in PA composition due to seasonal changes was significant but relatively small. Senecionine-like PAs were the dominant PAs in roots, while jacobine-/erucifoline-like PAs were dominant in shoots. The variation of PA concentration was significantly correlated with temperature, day length, and plant age. A correlation analysis showed that PAs were not reallocated when biomass was lost in winter. Overall, our study showed that PA composition of each genotype changed over seasons in a different manner but seasonal variation did not overrule the differences in PA composition among genotypes.

Despite the extensive studies on latex, some fundamental questions on their chemical specialization and the factors influencing this specialization have yet to be investigated. To address this issue, latexes and their bearing tissues from diverse species were profiled by 1HNMR and GC-MS. Additionally, the antiherbivory activity of these materials was tested against thrips (Frankliniella occidentalis Pergande, 1895). The multivariate data analysis showed a clear separation between latexes and leaves from the same species. Conversely, the chemical profiles of latexes from different species were highly similar, that is, they displayed much less metabolic species-specificity. These shared chemical profiles of latexes were reflected in their overall higher mortality index (80.4% ± 7.5) against thrips compared with their bearing tissues (55.5% ± 14.9). The metabolites correlated to the antiherbivory activity of latexes were triterpenoids and steroids. However, the activity could not be attributed to any single terpenoid. This discrepancy and the reduction of the latex activity after fractionation suggested a complementary effect of the compounds when in a mixture as represented by the latex. Additionally, aqueous fractions of several latexes were found to possess simple spectra, even with only 1 metabolite. These metabolites were determined to be organic acids that might be involved in the modulation of the rate of latex coagulation, potentially increasing the sealing and trapping effects of the latex.

Ultraviolet (UV) radiation has emerged as an environmental cue with potential uses to enhance plant protection against arthropod pests in agriculture. UV can augment constitutive and inducible plant defenses against herbivorous arthropods. Here we investigated whether application of supplemental UV to chrysanthemum (Chrysanthemum × morifolium Ramat) cuttings during their rooting phase enhanced plant resistance to an important insect pest, Western flower thrips (Frankliniella occidentalis). For this, we analyzed how several daily UV exposure times affected plant damage by thrips on three different chrysanthemum cultivars. The most effective UV dose and responsive cultivar were further used to determine the UV effects on host plant preference by thrips, leaf metabolome and the induction of jasmonic acid (JA)-associated defenses. Our results showed that while short UV daily exposure times increased chrysanthemum resistance to thrips, longer exposure times had the opposite effect. Furthermore, we showed that UV-mediated induction of chrysanthemum resistance to thrips was genotype dependent and can persist after the end of the of the UV treatment. Yet, this induction was not transferred to the next generation from mother plants to cuttings. Nontargeted metabolomic, enzymatic and hormone analyses further revealed that UV slightly affected the leaf metabolome of chrysanthemum plants, and it enhanced the induction of JA-associated signaling after thrips infestation. Taken together, our results suggest that supplemental UV might modulate both constitutive and inducible chrysanthemum defenses against thrips.

Western flower thrips (WFT) are a major pest on many crops, including tomato. Thrips cause yield losses, not only through feeding damage, but also by the transmission of viruses of which the Tomato Spotted Wilt Virus is the most important one. In cultivated tomato, genetic diversity is extremely low, and all commercial lines are susceptible to WFT. Several wild relatives are WFT resistant and these resistances are based on glandular trichome-derived traits. Introgression of these traits in cultivated lines did not lead to WFT resistant commercial varieties so far. In this study, we investigated WFT resistance in cultivated tomato using a F2 population derived from a cross between a WFT susceptible and a WFT resistant cultivated tomato line. We discovered that this WFT resistance is independent of glandular trichome density or trichome-derived volatile profiles and is associated with three QTLs on chromosomes 4, 5 and 10. Foliar metabolic profiles of F3 families with low and high WFT feeding damage were clearly different. We identified α-tomatine and a phenolic compound as potential defensive compounds. Their causality and interaction need further investigation. Because this study is based on cultivated tomato lines, our findings can directly be used in nowadays breeding programs.

A long‐standing question in ecology is how species interactions are structured within communities. Although evolutionary theory predicts close size matching between floral nectar tube depth and pollinator proboscis length of interacting species, such size matching has seldom been shown and explained in multispecies assemblages. Here, we investigated the degree of size matching among Asteraceae and their pollinators and its relationship with foraging efficiency. The majority of pollinators, especially Hymenoptera, choose plant species on which they had high foraging efficiencies. When proboscides were shorter than nectar tubes, foraging efficiency rapidly decreased because of increased handling time. When proboscides were longer than nectar tubes, a decreased nectar reward rather than an increased handling time made shallow flowers more inefficient to visit. Altogether, this led to close size matching. Overall, our results show the importance of nectar reward and handling time as drivers of plant–pollinator network structure.

Resins are one of the first sites of interaction between plants and biotic and abiotic factors. Despite their evident morphological and chemical differentiation from other plant organs, the detailed correlation between resins and biological or environmental factors is not yet clear. In this study, 1H nuclear magnetic resonance (NMR), gas chromatography coupled with mass spectrometry (GC-MS) and high-performance thin-layer chromatography (HPTLC)-based profiling techniques were applied to the metabolic characterisation of plant resins of different species and season of collection, using samples from five different species that were collected during early and late spring. The 1H NMR analysis confirmed the main metabolic groups in the resins to be terpenoids and further GC-MS analysis revealed a notable chemical variation between the species and collection periods. Abies grandis displayed a significant differentiation from the other species, showing a higher number of monoterpenes. The HPTLC-based profiling method hyphenated with multivariate data analysis (MVDA) also showed a clear separation confirming the GC-MS terpenoidal profiling results. Additionally, the unknown compounds were obtained by preparative TLC for identification. Based on the results of the three analytical platforms, it was concluded that the major difference in chemical composition of pine species was between species rather than the collection period. Nonetheless, the chemical profiles of resins from different species and collection periods can be well discriminated and correlated to mono- and sesquiterpenes in the case of species and diterpenes for the collection periods.

Summary In a study of Cynoglossum officinale L. it was found that the number of seeds per flower increased with plant weight, and that flower production was inversely related to the number of seeds per flower. As a result, the number of seeds and total seed weight increased proportionally with plant weight. Seed size was not related to plant weight. Differences in seed size within the individual were, in part, related to the position of the seed on the cyme and not related to the number of seeds within a flower. The relationship, between plant size and female fitness found in this study is discussed in relation to life-history theory.

International audience; We measured the relationship between selfing rates and flower number in an experimental population of bumblebee pollinated Cynoglossum officinale, with plants differing in flower number. Results were compared with the prediction of a model based on pollen dynamics and pollinator behaviour. The selfing rate, as measured by multilocus oligonucleotide DNA fingerprinting, increased with flower number and ranged from 0% to 70%. Flowers on large plants received an equal number of visits from bumblebees as flowers on small plants. On large plants more flowers in a row were visited, inducing geitonogamy. The overall relationship between selfing rate and number of flowers can be explained by pollen dynamics and pollinator behaviour without invoking postpollination processes such as differential pollen tube growth and abortion.

Understanding the mechanisms involved in host plant resistance opens the way for improved resistance breeding programs by using the traits involved as markers. Pest management is a major problem in cultivation of ornamentals. Gladiolus (Gladiolus hybridus L.) is an economically important ornamental in the Netherlands. Gladiolus is especially sensitive to attack by western flower thrips (Frankliniella occidentalis (Pergande) (Thysanoptera:Thripidae)). The objective of this study was, therefore, to investigate morphological and chemical markers for resistance breeding to western flower thrips in Gladiolus varieties. We measured thrips damage of 14 Gladiolus varieties in a whole-plant thrips bioassay and related this to morphological traits with a focus on papillae density. Moreover, we studied chemical host plant resistance to using an eco-metabolomic approach comparing the 1H NMR profiles of thrips resistant and susceptible varieties representing a broad range of papillae densities. Thrips damage varied strongly among varieties: the most susceptible variety showed 130 times more damage than the most resistant one. Varieties with low thrips damage had shorter mesophylls and epidermal cells, as well as a higher density of epicuticular papillae. All three traits related to thrips damage were highly correlated with each other. We observed a number of metabolites related to resistance against thrips: two unidentified triterpenoid saponins and the amino acids alanine and threonine. All these compounds were highly correlated amongst each other as well as to the density of papillae. These correlations suggest that papillae are involved in resistance to thrips by producing and/or storing compounds causing thrips resistance. Although it is not possible to distinguish the individual effects of morphological and chemical traits statistically, our results show that papillae density is an easy marker in Gladiolus-breeding programs targeted at increased resistance to thrips.

Fungal endophytes isolated from two latex bearing species were chosen as models to show their potential to expand their host plant chemical diversity. Thirty-three strains were isolated from Alstonia scholaris (Apocynaceae) and Euphorbia myrsinites (Euphorbiaceae). High performance thin layer chromatography (HPTLC) was used to metabolically profile samples. The selected strains were well clustered in three major groups by hierarchical clustering analysis (HCA) of the HPTLC data, and the chemical profiles were strongly correlated with the strains’ colony size. This correlation was confirmed by orthogonal partial least squares (OPLS) modeling using colony size as “Y” variable. Based on the multivariate data analysis of the HPTLC data, the fastest growing strains of each cluster were selected and used for subsequent experiments: co-culturing to investigate interactions between endophytes-phytopathogens, and biotransformation of plant metabolites by endophytes. The strains exhibited a high capacity to fight against fungal pathogens. Moreover, there was an increase in the antifungal activity after being fed with host-plant metabolites. These results suggest that endophytes play a role in plant defense mechanisms either directly or by biotransformation/induction of metabolites. Regarding HPTLC-based metabolomics, it has proved to be a robust approach to monitor the interactions among fungal endophytes, the host plant and potential phytopathogens.

Background and aimsMany plant species grow better in sterilized than in live soil. Foliar application of SA mitigates this negative effect of live soil on the growth of the plant Jacobaea vulgaris. To examine what causes the positive effect of SA application on plant growth in live soils, we analyzed the effects of SA application on the composition of active rhizosphere bacteria in the live soil. Methods We studied this over four consecutive plant cycles (generations), using mRNA sequencing of the microbial communities in the rhizosphere of J. vulgaris. ResultsOur study shows that the composition of the rhizosphere bacterial communities of J. vulgaris greatly differed among generations. Application of SA resulted in both increases and decreases in a number of active bacterial genera in the rhizosphere soil, but the genera that were affected by the treatment differed among generations. In the first generation, there were no genera that were significantly affected by the SA treatment, indicating that induction of the SA defense pathway in plants does not lead to immediate changes in the soil microbial community. 89 species out of the total 270 (32.4%) were present in all generations in all soils of SA-treated and control plants suggesting that these make up the “core” microbiome. On average in each generation, 72.9% of all genera were present in both soils. Application of SA to plants significantly up-regulated genera of Caballeronia, unclassified Cytophagaceae, Crinalium and Candidatus Thermofonsia Clade 2, and down-regulated genera of Thermomicrobiales, unclassified Rhodobacterales, Paracoccus and Flavihumibacter. While the functions of many of these bacteria are poorly understood, bacteria of the genus Caballeronia play an important role in fixing nitrogen and promoting plant growth, and hence this suggests that activation of the SA signaling pathway in J. vulgaris plants may select for bacterial genera that are beneficial to the plant. ConclusionsOverall, our study shows that aboveground activation of defenses in the plant affects soil microbial communities and, as soil microbes can greatly influence plant performance, this implies that induction of plant defenses can lead to complex above-belowground feedbacks. Further studies should examine how activation of the SA signaling pathway in the plant changes the functional genes of the rhizosphere soil bacterial community.

Phytohormone applications are used to mimic herbivory and can induce plant defences. This study investigated (i) metabolomic changes in leaf tissues of Jacobaea vulgaris and J. aquatica after methyl jasmonate (MeJA) and salicylic acid (SA) applications and (ii) the effects on a leaf-chewing, a leaf-mining and a piercing-sucking herbivore. MeJA treated leaves showed clearly different metabolomic profiles than control leaves, while the differences in metabolomic profiles between SA treated leaves and control leaves were less clear. More NMR peaks increased than decreased after MeJA treatment while this pattern was reversed after SA treatment. The leaf-chewing (Mamestra brassicae) and the leaf-mining herbivores (Liriomyza trifolii) fed less on MeJA-treated leaves compared to control and SA-treated leaves while they fed equally on the latter two. In J. aquatica but not in J. vulgaris, SA treatment reduced feeding damage by the piercing-sucking herbivore (Frankliniella occidentalis). Based on the herbivory and metabolomic data after phytohormone application, we made speculations as follows: For all three herbivore species, plants with high levels of threonine and citric acid showed less herbivory while plants with high levels of glucose showed more herbivory. Herbivory by thrips was lower on plants with high levels of alanine while it was higher on plants with high levels of 3,5-dicaffeoylquinic acid. The plant compounds that related to feeding of piercing-sucking herbivore were further verified with previous independent experiments.

Many plants produce exudates as defense mechanism to hamper pests and pathogens. Plant exudates include latexes, resins, gums, root exudates, and also biofluids produced by carnivorous plants. Plants exudates consist of, among others, polysaccharides, lipids, proteins, organic acids and bases, sugars, and amino acids. All these small molecules are NADES components. In this chapter, the chemical compositions of plant exudates, especially leaf exudates, and Drosera plant biofluids will be elaborated. Metabolic profiling approaches based on 1H NMR and GC–MS were performed on the biofluids of the Drosera species. The 1H NMR spectra showed a high level of myo-inositol and sugars in the biofluids as well as some organic acids. To further confirm the identity of the sugars and to identify some minor components, GC–MS was employed. This gave similar results as the 1H NMR spectroscopy. All the tested exudates were found to contain sugars, such as fructose, glucose, arabinose, sucrose, and xylose; organic acids, such as glucuronic acid and ascorbic acid; and myo-inositol. Those Drosera ingredients are known to be strong hydrogen-bond donors. Based on the information obtained from the 1H NMR and GC–MS results, new NADESs compositions were proposed and they were tested in vitro, of which indeed five combinations formed liquids. However, considering that these Drosera biofluids consist mainly of water and other materials, it may indicate that the stable high viscosity droplets typical for the Drosera species must be due in part to other physical-chemical forces, such as gel formation by polysaccharides with calcium ions. A possible role of the NADES-like liquids could be in stabilizing proteins, or the formation of biofilms enabling a faster digesting of the prey. The myo-inositol that was clearly a major compound in the exudate might be involved in protecting the leaves and the sugar containing exudate from herbivory. The occurrence of some common products found in fermentation processes might indicate that microorganisms are present in the biofluid.

It is increasingly evident that plants interact with their outside world through the production of volatile organic compounds,1-5 but whether the volatiles have evolved to serve in plant defense is still a topic of considerable debate.3,6-8 Unharmed leaves constitutively release small amounts of volatiles, but when the leaves are damaged by herbivorous arthropods, they emit substantially more volatiles. These herbivore-induced plant volatiles (HIPVs) attract parasitoids and predators that kill insect herbivores,9-12 and this can benefit the plants.13,14 As yet, however, there is no tangible evolutionary evidence that this tritrophic interplay contributes to the selection forces that have shaped the volatile emissions of plants.2,3,5-8,15 With this in mind, we investigated the evolutionary changes in volatile emissions in invasive common ragwort and the respective defensive roles of its constitutive and inducible volatiles. This Eurasian plant has invaded other continents, where it evolved for many generations in the absence of specialized herbivores and their natural enemies. We found that, compared to native ragworts, invasive plants release higher levels of constitutive volatiles but considerably lower levels of herbivore-induced volatiles. As a consequence, invasive ragwort is more attractive to a specialist moth but avoided by an unadapted generalist moth. Importantly, conforming to the indirect defense hypothesis, a specialist parasitoid was much more attracted to caterpillar-damaged native ragwort, which was reflected in higher parasitism rates in a field trial. The evolution of foliar volatile emissions appears to be indeed driven by their direct and indirect roles in defenses against insects.

Background Collectively, plants produce a huge variety of secondary metabolites (SMs) which are involved in the adaptation of plants to biotic and abiotic stresses. The most characteristic feature of SMs is their striking inter- and intraspecific chemical diversity. Cytochrome P450 monooxygenases (CYPs) often play an important role in the biosynthesis of SMs and thus in the evolution of chemical diversity. Here we studied the diversity and evolution of CYPs of two Jacobaea species which contain a characteristic group of SMs namely the pyrrolizidine alkaloids (PAs). Results We retrieved CYPs from RNA-seq data of J. vulgaris and J. aquatica, resulting in 221 and 157 full-length CYP genes, respectively. The analyses of conserved motifs confirmed that Jacobaea CYP proteins share conserved motifs including the heme-binding signature, the PERF motif, the K-helix and the I-helix. KEGG annotation revealed that the CYPs assigned as being SM metabolic pathway genes were all from the CYP71 clan but no CYPs were assigned as being involved in alkaloid pathways. Phylogenetic analyses of full-length CYPs were conducted for the six largest CYP families of Jacobaea (CYP71, CYP76, CYP706, CYP82, CYP93 and CYP72) and were compared with CYPs of two other members of the Asteraceae, Helianthus annuus and Lactuca sativa, and with Arabidopsis thaliana. The phylogenetic trees showed strong lineage specific diversification of CYPs, implying that the evolution of CYPs has been very fast even within the Asteraceae family. Only in the closely related species J. vulgaris and J. aquatica, CYPs were found often in pairs, confirming a close relationship in the evolutionary history. Conclusions This study discovered 378 full-length CYPs in Jacobaea species, which can be used for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.

Based on the hypothesis that variation of the metabolomes of latex is a response to selective pressure and should thus be affected differently from other organs, their variation could provide insight into the defensive chemical selection of plants. Metabolic profiling was utilized to compare tissues of Euphorbia species collected in various regions. The metabolic variation of latexes was much more limited than that of other organs. In all of the species, the levels of polyisoprenes and terpenoids were found to be much higher in latexes than in leaves and roots. Polyisoprenes were also observed to physically delay the contact and growth of pathogens with plant tissues. A secondary barrier composed of terpenes and, in particular, 24-methylenecycloartanol, exhibited antifungal activity. These results, together with the known roles of the enzymes also present in latexes, demonstrate that they are part of a cooperative defense system that comprises both biochemical and physical elements.

Induction of defenses is one of the most widely accepted eco-friendly approaches for management of pests and diseases. Seeds are receptive to resistance-inducing chemicals and could offer broad-spectrum protection at the early stages of development. However, seed treatment with elicitors has previously been shown to differentially influence induced defense responses among cultivars and thus, could hamper commercial exploitation. In this context, the objective of the present study was to evaluate the genotype-dependent ability of jasmonic acid (JA) to induce resistance against western flower thrips (WFT) at the seed stage. We examined the variation in inducibility of resistance in eight commercial tomato cultivars. Causal factors accounting for discrepancies in JA-induced responses at the seed stage were phenotypically and biochemically evaluated. Seed receptivity to exogenous JA appeared to be cultivar dependent. Thrips associated silver damage was only reduced in JA seed-treated plants of cultivar Carousel. Enhancement of resistance, was not associated with activation of defense-related traits such as polyphenol oxidase activity (PPO), trichomes or volatiles. Sulfuric acid scarification, prior to JA seed incubation, significantly augmented the embryonic responsiveness to JA in cv. Moneymaker without an adverse effect on growth. Hence, these results support the hypothesis that seed coat permeability is a key factor for successfully inducing JA mediated thrips defenses. The outcome of our study is of translational value as it creates opportunities for the seed industry to perform pre-treatments on non-responsive cultivars as well as for tomato breeding programs to select for genetic traits that affect seed permeability.

Auxins are commonly used for commercial propagation of chrysanthemums by stem cuttings. Recent studies imply that these root-promoting hormones also affect plant defense responses. The underlying motive of this study stems from the serendipitous observation that water dipping of auxin-coated cuttings beneficially affected thrips herbivory. Therefore, the primary objective of this investigation was to explore the role of indole-3-butyric acid (IBA) in relation to herbivore susceptibility in chrysanthemum. We observed contrasting findings concerning the physical presence of IBA and it&rsquo, s role in promoting susceptibility of cuttings to thrips, which may in part be explained by the phenotypical variations of cuttings generated from mother plants. Nonetheless, we repeatedly demonstrated considerable protection, in some experiments up to 37%, against thrips and leaf miner upon water dipping of IBA-coated cuttings. Assessment of polyphenol oxidase activity (PPO), 14 days after dipping treatment, suggests that neither direct induction nor priming of plant defenses are involved. Future experiments aimed at understanding the early signaling events may help to explain the underlying mechanisms involved in conferring herbivore protection. We propose a dual role for auxins in early integrated pest management strategies to maximize plant development and minimize herbivory through feasible, cost-effective water dipping treatments.

Supplemental ultraviolet radiation increases tomato resistance to thrips herbivory through activation of jasmonic acid signaling, and without changes in the plant metabolome or the density of type-VI trichomes and their associated volatiles., Ultraviolet (UV) radiation can modulate plant defenses against herbivorous arthropods. We investigated how different UV exposure times and irradiance intensities affected tomato (Solanum lycopersicum) resistance to thrips (Frankliniella occidentalis) by assessing UV effects on thrips-associated damage and host-selection, selected metabolite and phytohormone contents, expression of defense-related genes, and trichome density and chemistry, the latter having dual roles in defense and UV protection. Short UV daily exposure times increased thrips resistance in the cultivar ‘Moneymaker’ but this could not be explained by changes in the contents of selected leaf polyphenols or terpenes, nor by trichome-associated defenses. UV irradiance intensity also affected resistance to thrips. Further analyses using the tomato mutants def-1, impaired in jasmonic acid (JA) biosynthesis, od-2, defective in the production of functional type-VI trichomes, and their wild-type, ‘Castlemart’, showed that UV enhanced thrips resistance in Moneymaker and od-2, but not in def-1 and Castlemart. UV increased salicylic acid (SA) and JA-isoleucine concentrations, and increased expression of SA- and JA-associated genes in Moneymaker, while inducing expression of JA-defensive genes in od-2. Our results demonstrate that UV-mediated enhancement of tomato resistance to thrips is probably associated with the activation of JA-associated signaling, but not with plant secondary metabolism or trichome-related traits.

Plants produce an extremely diverse array of metabolites that mediate many aspects of plant-environment interactions. In the context of plant-herbivore interactions, it is as yet poorly understood how natural backgrounds shape the bioactivity of individual metabolites. We tested the effects of a methanol extract of Jacobaea plants and five fractions derived from this extract, on survival of western flower thrips (WFT). When added to an artificial diet, the five fractions all resulted in a higher WFT survival rate than the methanol extract. In addition, their expected combined effect on survival, assuming no interaction between them, was lower than that of the methanol extract. The bioactivity was restored when the fractions were combined again in their original proportion. These results strongly suggest synergistic interactions among the fractions on WFT survival rates. We then tested the effects of two pyrrolizidine alkaloids (PAs), free base retrorsine and retrorsine N-oxide, alone and in combination with the five shoot fractions on WFT survival. The magnitude of the effects of the two PAs depended on the fraction to which they were added. In general, free base retrorsine was more potent than retrorsine N-oxide, but this was contingent on the fraction to which these compounds were added. Our results support the commonly held, though seldom tested, notion that the efficacy of plant metabolites with respect to plant defence is dependent on their phytochemical background. It also shows that the assessment of bioactivity cannot be decoupled from the natural chemical background in which these metabolites occur.

Western flower thrips (WFT) is one of the most economically important pest insects of many crops worldwide. Recent EU legislation has caused a dramatic shift in pest management strategies, pushing for tactics that are less reliable on chemicals. The development of alternative strategies is therefore an issue of increasing urgency. This paper reviews the main control tactics in integrated pest management (IPM) of WFT, with the focus on biological control and host plant resistance as areas of major progress. Knowledge gaps are identified and innovative approaches emphasised, highlighting the advances in 'omics' technologies. Successful programmes are most likely generated when preventive and therapeutic strategies with mutually beneficial, cost-effective and environmentally sound foundations are incorporated. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Plant defenses inducible by herbivorous arthropods can determine performance of subsequent feeding herbivores. We investigated how infestation of tomato (Solanum lycopersicum) plants with the Western flower thrips (Frankliniella occidentalis) alters host plant suitability and foraging decisions of their conspecifics. We explored the role of delayed-induced jasmonic acid (JA)-mediated plant defense responses in thrips preference by using the tomato mutant def-1, impaired in JA biosynthesis. In particular, we investigated the effect of thrips infestation on trichome-associated tomato defenses. The results showed that when offered a choice, thrips preferred non-infested plants over infested wild-type plants, while no differences were observed in def-1. Exogenous application of methyl jasmonate restored the repellency effect in def-1. Gene expression analysis showed induction of the JA defense signaling pathway in wild-type plants, while activating the ethylene signaling pathway in both genotypes. Activation of JA defenses led to increases in type-VI leaf glandular trichome densities in the wild type, augmenting the production of trichome-associated volatiles, i.e. terpenes. Our study revealed that plant-mediated intraspecific interactions between thrips are determined by JA-mediated defenses in tomato. We report that insects can alter not only trichome densities but also the allelochemicals produced therein, and that this response might depend on the magnitude and/or type of the induction.

Specialist herbivores feed on a restricted number of related plant species and may suffer food shortage if overexploitation leads to periodic defoliation of their food plants. The density, size and quality of food plants are important factors that determine the host plant choice of specialist herbivores. To explore how all these factors influence their oviposition behaviour, we used the cinnabar moth Tyria jacobaeae and the hybrids of a cross between Jacobaea vulgaris and J. aquatica as a study system. While defoliation by the cinnabar moth is common in the coastal area of The Netherlands, it is relatively rare in inland ragwort population. Ragworts contain pyrrolizidine alkaloids (PAs) and those that are found in coastal areas are rich in jacobine-like PAs while those that occur inland are rich in erucifoline-like PAs. We tested how the oviposition preference was influenced by plant size, nitrogen and water content and PA composition. We used cinnabar moth populations from a regularly defoliated area, Meijendel, and Bertogne, a rarely defoliated area. Our results revealed no effects of nitrogen or water content on oviposition preference. Moths from both populations laid larger egg batches on the plants rich in jacobine-like PAs. Moths from Meijendel preferred larger plants and spread their eggs over more egg batches that were, on average, smaller than those of Bertogne moths. These results suggest that Meijendel moths adopted a oviposition strategy to cope with potential defoliation.

Historically, latex-bearing plants have been regarded as important medicinal resources in many countries due to their characteristic latex ingredients. They have also often been endowed with a social or cultural significance in religious or cult rituals or for hunting. Initial chemical studies focused on the protein or peptide content but recently the interest extended to smaller molecules. Latex has been found to contain a broad range of specialized metabolites such as terpenoids, cardenolides, alkaloids, and phenolics, which are partly responsible for their antibacterial, antifungal, anthelmintic, cytotoxic, and insect-repellent activities. The diversity in biology and chemistry of latexes is supposedly associated to their ecological roles in interactions with exogenous factors. Latexes contain unique compounds that are different to those found in their bearing plants. Exploring the feasibility of plant latex as a new type of bioactive chemical resource, this review paper covers the chemical characterization of plant latexes, extending this to various other plant exudates. Also, the factors influencing this chemical differentiation and the production, transportation, and chemistry of the latex exudates are described, based on ecological and biochemical mechanisms. We also proposed a latex coagulation model involving 4 general conserved steps. Therefore, the inherent defensive origin of latexes is recognized as their most valuable character and encourages one to pay attention to these materials as alternative sources to discover metabolites with insecticidal or antimicrobial activity.

In this study we investigated the effect of methyl jasmonate (MeJA) application on pyrrolizidine alkaloid (PA) concentration and composition of two closely related Jacobaea species. In addition, we examined whether MeJA application affected herbivory of the polyphagous leaf feeding herbivore Spodoptera exigua. A range of concentrations of MeJA was added to the medium of Jacobaea vulgaris and J. aquatica tissue culture plants grown under axenic conditions. PA concentrations were measured in roots and shoots using LC-MS/MS. In neither species MeJA application did affect the total PA concentration at the whole plant level. In J. vulgaris the total PA concentration decreased in roots but increased in shoots. In J. aquatica a similar non-significant trend was observed. In both Jacobaea species MeJA application induced a strong shift from senecionine- to erucifoline-like PAs, while the jacobine- and otosenine-like PAs remained largely unaffected. The results show that MeJA application does not necessarily elicits de novo synthesis, but rather leads to PA conversion combined with reallocation of certain PAs from roots to shoots. S. exigua preferred feeding on control leaves of J. aquatica over MeJA treated leaves, while for J. vulgaris both the control and MeJA treated leaves were hardly eaten. This suggests that the MeJA-induced increase of erucifoline-like PAs can play a role in resistance of J. aquatica to S. exigua. In J. vulgaris resistance to S. exigua may already be high due to the presence of jacobine-like PAs or other resistance factors.

Ultraviolet radiation (UV) is an important modulator of plant defenses against biotic stresses. We have recently described that different supplemental UV exposure times and irradiance intensities enhanced tomato (Solanum lycopersicum) resistance to Western flower thrips (Frankliniella occidentalis). UV increased jasmonic acid-isoleucine (JA-Ile) and salicylic acid (SA) levels, as well as the expression of JA- and SA-responsive genes, before thrips herbivory. Here we report how UV affects tomato defense responses upon thrips infestation, and resistance to pathogens that are susceptible to the activation of SA-associated defenses. Our experiments reveal that, at 7 days after thrips infestation, UV did not enhance the levels of jasmonates, auxin or abscisic acid. UV also did not affect the expression of JA-responsive genes in the cultivar Moneymaker, the jasmonate deficient mutant def-1, the type-VI trichome deficient mutant od-2, or their wild-type Castlemart. However, UV strongly activated SA-associated defense responses in def-1 after thrips infestation. Further bioassays showed that UV increased def-1 resistance to the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000, which is susceptible to SA-mediated defenses. Our results suggest that UV might enhance tomato resistance to this pathogen in the JA deficient genotype through the activation of SA defenses.

We hypothesized that due to the absence of specialist herbivores in introduced ranges, invasive plants have evolved decreased allocation to carbohydrate storage for regrowth ability and as a consequence allocate more to growth. In this study, we compared plant growth, carbohydrate storage and regrowth ability of invasive and native Jacobaea vulgaris in response to complete shoot defoliation. We used invasive J. vulgaris genotypes from three geographically and climactically distinct regions and compared these with native genotypes from Europe. We found that invasive genotypes initially grew larger while native genotypes regrew larger after defoliation. Before defoliation, the carbohydrate storage in roots of invasive genotypes was 38% lower than native genotypes. Biomass after regrowth increased with root carbohydrate storage while it decreased with structural root mass, showing that it is crucial to study root storage and structural components separately in order to investigate plant regrowth. All studied traits of invasive populations from the three geographically and climatologically distinct regions changed in the same expected direction suggesting that the shifts in herbivore guild were causal to the observed change in growth and regrowth ability rather than environmental factors.

Western flower thrips (WFT) Frankliniella occidentalis (Pergande) is a key agricultural pest of cultivated tomatoes. Induced host plant resistance by activating jasmonic acid (JA) signaling pathway constitutes a promising method for WFT control. The phytotoxin coronatine (COR), produced by Pseudomonas syringae pv. tomato DC3000 (Pst), mimics the plant hormone JA-Isoleucine and can promote resistance against herbivorous arthropods. Here we determined the effect of Pst and COR on tomato resistance against WFT, induction of JA and salicylic acid (SA) associated defenses, and plant chemistry. Additionally, we investigated the presence of other components in Pst-derived and filtered culture medium, and their interactive effect with COR on tomato resistance to WFT. Our results showed that infiltration of COR or Pst reduced WFT feeding damage in tomato plants. COR and Pst induced the expression of JA-associated gene and protein marker. COR also induced expression of a SA-related responsive gene, although at much less magnitude. Activation of JA defenses in COR and Pst infiltrated plants did not affect density of type VI leaf trichomes, which are defenses reported to be induced by JA. An untargeted metabolomic analysis showed that both treatments induced strong changes in infiltrated leaves, but leaf responses to COR or Pst slightly differed. Application of the Pst-derived and filtered culture medium, containing COR but not viable Pst, also increased tomato resistance against WFT confirming that the induction of tomato defenses does not require a living Pst population to be present in the plant. Infiltration of tomato plants with low concentrations of COR in diluted Pst-derived and filtered culture medium reduced WFT feeding damage in a greater magnitude than infiltration with an equivalent amount of pure COR indicating that other elicitors are present in the medium. This was confirmed by the fact that the medium from a COR-mutant of Pst also strongly reduced silver damage. In conclusion, our results indicate that induction of JA defenses by COR, Pst infection, the medium of Pst and the medium of a Pst COR- mutant increased resistance against WFT. This was not mediated by the reinforcement of leaf trichome densities, but rather the induction of chemical defenses.

In cultivated tomato (Solanum lycopersicum), increases in photosynthetically active radiation (PAR) induces type VI leaf glandular trichomes, which are important defensive structures against arthropod herbivores. Yet, how PAR affects the type VI trichome-associated leaf chemistry and its biological significance with respect to other photomorphogenic responses in this agronomically important plant species is unknown. We used the type VI trichome deficient tomato mutant odorless-2 (od-2) and its wild-type to investigate the influence of PAR on trichome-associated chemical defenses against thrips (Frankliniella occidentalis). High PAR increased thrips resistance in wild-type plants, but not in od-2. Furthermore, under high PAR thrips preferred od-2 over the wild-type. Both genotypes increased type VI trichome densities under high PAR. Wild-type plants, however, produced more trichome-associated allelochemicals, i.e. terpenes and phenolics, these being undetectable or barely altered in od-2. High PAR increased leaf number and thickness, and induced profound but similar metabolomic changes in wild-type and od-2 leaves. Enhanced PAR also increased levels of abscisic acid in wild-type and od-2 plants, and of auxin in od-2, while the salicylic acid and jasmonates concentrations were unaltered. However, in both genotypes, high PAR induced the expression of jasmonic acid (JA)-responsive defense-related genes. Taken together, our results demonstrate that high PAR-mediated induction of trichome-associated chemical defenses plays a prominent role in tomato-thrips interactions.

Variation in the induction of plant defenses along the plant canopy can determine distribution and colonization of arthropod herbivores within the plant. In tomato, type VI glandular trichomes, which are epidermal defensive structures, and their derived volatiles are induced by the phytohormone jasmonic acid (JA). How JA-mediated induction of these trichome-associated chemical defenses depends on the leaf developmental stage and correlates with resistance against herbivory is unknown. We showed that application of JA reduced thrips-associated damage, however the amplitude of this response was reduced in the fully developed leaves compared to those still developing. Although JA increased type-VI trichome densities in all leaf developmental stages, as well as JA-inducible defensive proteins, these increases were stronger in developing leaves. Remarkably, the concentration of trichome-derived volatiles was induced by JA to a larger degree in developing leaves than in fully developed leaves. In fully developed leaves, the increase in trichome-derived volatiles was explained by an enhanced production per trichome, while in developing leaves this was mainly caused by increases in type-VI trichome densities. Together, we showed that JA-mediated induction of trichome density and chemistry depends on leaf development stage, and it might explain the degree of thrips-associated leaf damage in tomato

The high structural diversity of plant metabolites suggests that interactions among them should be common. We investigated the effects of single metabolites and combinations of plant metabolites on insect herbivores. In particular we studied the interacting effects of pyrrolizidine alkaloid (PAs), and chlorogenic acid (CGA), on a generalist herbivore, Frankliniella occidentalis. We studied both the predominantly occurring PA N-oxides and the less frequent PA free bases. We found antagonistic effects between CGA and PA free bases on thrips mortality. In contrast PA N-oxides showed synergistic interactions with CGA. PA free bases caused a higher thrips mortality than PA N-oxides while the reverse was through for PAs in combination with CGA. Our results provide an explanation for the predominate storage of PA N-oxides in plants. We propose that antagonistic interactions represent a constraint on the accumulation of plant metabolites, as we found here for Jacobaea vulgaris. The results show that the bioactivity of a given metabolite is not merely dependent upon the amount and chemical structure of that metabolite, but also on the co-occurrence metabolites in, e.g., plant cells, tissues and organs. The significance of this study is beyond the concerns of the two specific groups tested here. The current study is one of the few studies so far that experimentally support the general conception that the interactions among plant metabolites are of great importance to plant-environment interactions.

We studied the impact of community diversity on the selection of bacterial communities in the rhizosphere by comparing the composition and the functional traits of these communities in soil and rhizosphere. Differences in diversity were established by inoculating into sterilized soils diluted suspensions of the same soil. We used 16S ribosomal RNA amplicon sequencing to determine the taxonomical structure of the bacterial communities and a shotgun metagenomics approach to investigate the potential functional diversity of the communities. By comparing the bacterial communities in soil and rhizosphere, the selective power of the plant was observed both at the taxonomic and functional level, although the diversity indices of soil and rhizosphere samples showed a highly variable, irregular pattern. Lesser variation, that is, more homogenization, was found for both the taxonomic structure and the functional profile of the rhizosphere communities as compared to the communities of the bulk soil. Network analysis revealed stronger interactions among bacterial operational taxonomic units in the rhizosphere than in the soil. The enrichment processes in the rhizosphere selected microbes with particular functional genes related to transporters, the Embden-Meyerhof-Parnas pathway and hydrogen metabolism. This selection was not random across bacteria with these functional traits, but it was species specific. Overall, this suggests that functional traits are a key to the assembly of bacterial rhizosphere communities.The ISME Journal advance online publication, 2 August 2016; doi:10.1038/ismej.2016.108.

Ultraviolet-B (UV-B) light plays a crucial role in plant–herbivorous arthropods interactions by inducing changes in constitutive and inducible plant defenses. In particular, constitutive defenses can be modulated by UV-B-induced photomorphogenic responses and changes in the plant metabolome. In accordance, the prospective use of UV-B light as a tool to increase plant protection in agricultural practice has gained increasing interest. Changes in the environmental conditions might, however, modulate the UV-B -induced plant responses. While in some cases plant responses to UV-B can increase adaptation to changes in certain abiotic factors, UV-B-induced responses might be also antagonized by the changing environment. The outcome of these interactions might have a great influence on how plants interact with their enemies, e.g., herbivorous arthropods. Here, we provide a review on the interactive effects of UV-B and light quantity and quality, increased temperature and drought stress on plant biochemistry, and we discuss the implications of the outcome of these interactions for plant resistance to arthropod pests.