Aboveground and belowground herbivore species modify plant defense responses differently. Simultaneous attack can lead to non-additive effects on primary and secondary metabolite composition in roots and shoots. We previously found that aphid (Brevicoryne brassicae) population growth on Brassica oleracea was reduced on plants that were infested with nematodes (Heterodera schachtii) prior (4 weeks) to aphid infestation. Here, we examined how infection with root-feeding nematodes affected primary and secondary metabolites in the host plant and whether this could explain the increase in aphid doubling time from 3.8 to 6.7 days. We hypothesized that the effects of herbivores on plant metabolites would depend on the presence of the other herbivore and that nematode-induced changes in primary metabolites would correlate with reduced aphid performance. Total glucosinolate concentration in the leaves was not affected by nematode presence, but the composition of glucosinolates shifted, as gluconapin concentrations were reduced, while gluconapoleiferin concentrations increased in plants exposed to nematodes. Aphid presence increased 4-methoxyglucobrassicin concentrations in leaves, which correlated positively with the number of aphids per plant. Nematodes decreased amino acid and sugar concentrations in the phloem. Aphid population doubling time correlated negatively with amino acids and glucosinolate levels in leaves, whereas these correlations were non-significant when nematodes were present. In conclusion, the effects of an herbivore on plant metabolites were independent of the presence of another herbivore. Nematode presence reduced aphid population growth and disturbed feeding relations between plants and aphids.
Electronic supplementary material
The online version of this article (doi:10.1007/s10886-013-0338-4) contains supplementary material, which is available to authorized users.
Heterodera; Brevicoryne; Brassica; Glucosinolates; Phloem; EDTA
Insects and nematodes are the most diverse and abundant groups of multicellular animals feeding on plants on either side of the soil–air interface. Several herbivore-induced responses are systemic, and hence can influence the preference and performance of organisms in other plant organs. Recent studies show that plants mediate interactions between belowground plant parasitic nematodes (PPNs) and aboveground herbivorous insects. Based on the knowledge of plant responses to pathogens, we review the emerging insights on plant systemic responses against root-feeding nematodes and shoot-feeding insects. We discuss the potential mechanisms of plant-mediated indirect interactions between both groups of organisms and point to gaps in our knowledge. Root-feeding nematodes can positively or negatively affect shoot herbivorous insects, and vice versa. The outcomes of the interactions between these spatially separated herbivore communities appear to be influenced by the feeding strategy of the nematodes and the insects, as well as by host plant susceptibility to both herbivores. The potential mechanisms for these interactions include systemic induced plant defense, interference with the translocation and dynamics of locally induced secondary metabolites, and reallocation of plant nutritional reserves. During evolution, PPNs as well as herbivorous insects have acquired effectors that modify plant defense responses and resource allocation patterns to their advantage. However, it is also known that plants under herbivore attack change the allocation of their resources, e.g., for compensatory growth responses, which may affect the performance of other organisms feeding on the plant. Studying the chemical and molecular basis of these interactions will reveal the molecular mechanisms that are involved. Moreover, it will lead to a better understanding of the ecological relevance of aboveground–belowground interactions, as well as support the development of sustainable pest management technologies.
aboveground–belowground interactions; signaling interactions; systemic induced plant defense; secondary plant compounds; herbivores
Root herbivores are notoriously difficult to study, as they feed hidden in the soil. However, root herbivores may be traced by analyzing specific volatile organic compounds (VOCs) that are produced by damaged roots. These VOCs not only support parasitoids in the localization of their host, but also may help scientists study belowground plant-herbivore interactions. Herbivore-induced VOCs are usually analyzed by gas-chromatography mass spectrometry (GC-MS), but with this off-line method, the gases of interest need to be preconcentrated, and destructive sampling is required to assess the level of damage to the roots. In contrast to this, proton-transfer-reaction mass spectrometry (PTR-MS) is a very sensitive on-line, non-invasive method. PTR-MS already has been successfully applied to analyze VOCs produced by aboveground (infested) plant parts. In this review, we provide a brief overview of PTR-MS and illustrate how this technology can be applied to detect specific root-herbivore induced VOCs from Brassica plants. We also specify the advantages and disadvantages of PTR-MS analyses and new technological developments to overcome their limitations.
Chemical ecology; Root herbivory; Trace gas analysis; Induced indirect defense; Mass spectrometry; Volatile organic compound (VOC)
Plant pathogens and insect herbivores are prone to share hosts under natural conditions. Consequently, pathogen-induced changes in the host plant can affect herbivory, and vice versa. Even though plant viruses are ubiquitous in the field, little is known about plant-mediated interactions between viruses and non-vectoring herbivores. We investigated the effects of virus infection on subsequent infestation by a non-vectoring herbivore in a natural genotype of Trifolium repens (white clover). We tested whether infection with White clover mosaic virus (WClMV) alters (1) the effects of fungus gnat feeding on plant growth, (2) the attractiveness of white clover for adult fungus gnat females, and (3) the volatile emission of white clover plants. We observed only marginal effects of WClMV infection on the interaction between fungus gnat larvae and white clover. However, adult fungus gnat females clearly preferred non-infected over WClMV-infected plants. Non-infected and virus-infected plants could easily be discriminated based on their volatile blends, suggesting that the preference of fungus gnats for non-infected plants may be mediated by virus-induced changes in volatile emissions. The compound β-caryophyllene was exclusively detected in the headspace of virus-infected plants and may hence be particularly important for the preference of fungus gnat females. Our results demonstrate that WClMV infection can decrease the attractiveness of white clover plants for fungus gnat females. This suggests that virus infections may contribute to protecting their hosts by decreasing herbivore infestation rates. Consequently, it is conceivable that viruses play a more beneficial role in plant–herbivore interactions than generally thought.
Electronic supplementary material
The online version of this article (doi:10.1007/s00442-012-2322-z) contains supplementary material, which is available to authorized users.
Pathogens; Three-way interactions; Trifolium repens; Volatiles; White clover mosaic virus
Proton Transfer Reaction-Mass Spectrometry (PTR-MS) analyses revealed that damaged Brassica roots emit sulfur-containing volatiles. B. nigra, B. juncea and B. napus emitted isothiocyanate markers, whereas B. rapa, B. oleracea, and B. carinata emitted methanethiol. These compounds can be used as markers for root damage by insect larvae and other below-ground herbivores.
Background and aims
Plants damaged by herbivores emit a variety of volatile organic compounds (VOCs). Here we used proton-transfer reaction mass spectrometry (PTR-MS) as a sensitive detection method for online analysis of herbivore-induced VOCs. Previously, it was found that Brassica nigra plants emit several sulfur-containing VOCs when attacked by cabbage root fly (Delia radicum) larvae with m/z 60 as a marker for the formation of allylisothiocyanate from the glucosinolate sinigrin. We tested the hypothesis that m/z 60 emission occurs only in plants with sinigrin in their roots. Additionally, we tested the hypothesis that methanethiol, dimethylsulfide and dimethyldisulfide are only emitted after larval infestation.
Proton-transfer reaction mass spectrometry was used to track sulfur-containing VOCs from six different species of Brassica over time. The roots were either artificially damaged or infested with cabbage root fly larvae. Glucosinolate profiles of the roots were analysed using high-pressure liquid chromatography and compared with VOC emissions.
Brassica nigra, B. juncea and B. napus primarily emitted m/z 60 directly after artificial damage or root fly infestation. Sulfide and methanethiol emissions from B. nigra and B. juncea also increased after larval damage but much later (6–12 h after damage). Brassica rapa, B. oleracea and B. carinata principally emitted methanethiol after artificial and after larval damage. Brassica oleracea and B. carinata showed some increase in m/z 60 emission after larval damage. Comparison with root glucosinolate profiles revealed that sinigrin cannot be the only precursor for m/z 60.
The principal compound emitted after root damage is determined by the plant species, and not by damage type or root glucosinolate composition. Once determined, the principal compounds may be used as markers for identifying damaged or infested plants. Further analyses of plant enzymes involved in the breakdown of sulfur compounds is needed to reveal the origin of sulfur-containing VOCs from plants.
Plants attacked by herbivorous insects emit volatile organic compounds that are used by natural enemies to locate their host or prey. The composition of the blend is often complex and specific. It may vary qualitatively and quantitatively according to plant and herbivore species, thus providing specific information for carnivorous arthropods. Most studies have focused on simple interactions that involve one species per trophic level, and typically have investigated the aboveground parts of plants. These investigations need to be extended to more complex networks that involve multiple herbivory above- and belowground. A previous study examined whether the presence of the leaf herbivore Pieris brassicae on turnip plants (Brassica rapa subsp. rapa) influences the response of Trybliographa rapae, a specialist parasitoid of the root feeder Delia radicum. It showed that the parasitoid was not attracted by volatiles emitted by plants under simultaneous attack. Here, we analyzed differences in the herbivore induced plant volatile (HIPV) mixtures that emanate from such infested plants by using Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA). This multivariate model focuses on the differences between odor blends, and highlights the relative importance of each compound in an HIPV blend. Dual infestation resulted in several HIPVs that were present in both isolated infestation types. However, HIPVs collected from simultaneously infested plants were not the simple combination of volatiles from isolated forms of above- and belowground herbivory. Only a few specific compounds characterized the odor blend of each type of damaged plant. Indeed, some compounds were specifically induced by root herbivory (4-methyltridecane and salicylaldehyde) or shoot herbivory (methylsalicylate), whereas hexylacetate, a green leaf volatile, was specifically induced after dual herbivory. It remains to be determined whether or not these minor quantitative variations, within the background of more commonly induced odors, are involved in the reduced attraction of the root feeder’s parasitoid. The mechanisms involved in the specific modification of the odor blends emitted by dual infested turnip plants are discussed in the light of interferences between biosynthetic pathways linked to plant responses to shoot or root herbivory.
Plant–insect interactions; Multiple herbivory; Volatile organic compounds; Parasitoid; Trybliographa rapae; Delia radicum; Pieris brassicae; Brassicaceae
Plant defenses against insect herbivores and necrotrophic pathogens are differentially regulated by different branches of the jasmonic acid (JA) signaling pathway. In Arabidopsis, the basic helix-loop-helix leucine zipper transcription factor (TF) MYC2 and the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain TF ORA59 antagonistically control these distinct branches of the JA pathway. Feeding by larvae of the specialist insect herbivore Pieris rapae activated MYC2 transcription and stimulated expression of the MYC2-branch marker gene VSP2, while it suppressed transcription of ORA59 and the ERF-branch marker gene PDF1.2. Mutant jin1 and jar1-1 plants, which are impaired in the MYC2-branch of the JA pathway, displayed a strongly enhanced expression of both ORA59 and PDF1.2 upon herbivory, indicating that in wild-type plants the MYC2-branch is prioritized over the ERF-branch during insect feeding. Weight gain of P. rapae larvae in a no-choice setup was not significantly affected, but in a two-choice setup the larvae consistently preferred jin1 and jar1-1 plants, in which the ERF-branch was activated, over wild-type Col-0 plants, in which the MYC2-branch was induced. In MYC2- and ORA59-impaired jin1-1/RNAi-ORA59 plants this preference was lost, while in ORA59-overexpressing 35S:ORA59 plants it was gained, suggesting that the herbivores were stimulated to feed from plants that expressed the ERF-branch rather than that they were deterred by plants that expressed the MYC2-branch. The feeding preference of the P. rapae larvae could not be linked to changes in glucosinolate levels. Interestingly, application of larval oral secretion into wounded leaf tissue stimulated the ERF-branch of the JA pathway, suggesting that compounds in the oral secretion have the potential to manipulate the plant response toward the caterpillar-preferred ERF-regulated branch of the JA response. Our results suggest that by activating the MYC2-branch of the JA pathway, plants prevent stimulation of the ERF-branch by the herbivore, thereby becoming less attractive to the attacker.
plant defense signaling; jasmonic acid; plant–insect interactions; hormone crosstalk; Pieris rapae
The effect of direct chemical defences in plants on the performance of insect herbivores and their natural enemies has received increasing attention over the past 10 years. However, much less is known about the scale at which this variation is generated and maintained, both within and across populations of the same plant species. This study compares growth and development of the large cabbage butterfly, Pieris brassicae, and its gregarious pupal parasitoid, Pteromalus puparum, on three wild populations [Kimmeridge (KIM), Old Harry (OH) and Winspit (WIN)] and two cultivars [Stonehead (ST), and Cyrus (CYR)] of cabbage, Brassica oleracea. The wild populations originate from the coast of Dorset, UK, but grow in close proximity with one another. Insect performance and chemical profiles were made from every plant used in the experiment. Foliar glucosinolates (GS) concentrations were highest in the wild plants in rank order WIN > OH > KIM, with lower levels found in the cultivars. Caterpillar-damaged leaves in the wild cabbages also had higher GS levels than undamaged leaves. Pupal mass in P. brassicae varied significantly among populations of B. oleracea. Moreover, development time in the host and parasitoid were correlated, even though these stages are temporally separated. Parasitoid adult dry mass closely approximated the development of its host. Multivariate statistics revealed a correlation between pupal mass and development time of P. brassicae and foliar GS chemistry, of which levels of neoglucobrassicin appeared to be the most important. Our results show that there is considerable variation in quantitative aspects of defensive chemistry in wild cabbage plants that is maintained at very small spatial scales in nature. Moreover, the performance of the herbivore and its parasitoid were both affected by differences in plant quality.
Electronic supplementary material
The online version of this article (doi:10.1007/s00442-010-1861-4) contains supplementary material, which is available to authorized users.
Pieris brassicae; Pteromalus puparum; Glucosinolates; Allelochemical; Secondary plant chemistry
Plants under attack by aboveground herbivores emit complex blends of volatile organic compounds (VOCs). Specific compounds in these blends are used by parasitic wasps to find their hosts. Belowground induction causes shifts in the composition of aboveground induced VOC blends, which affect the preference of parasitic wasps. To identify which of the many volatiles in the complex VOC blends may explain parasitoid preference poses a challenge to ecologists. Here, we present a case study in which we use a novel bioinformatics approach to identify biologically relevant differences between VOC blends of feral cabbage (Brassica oleracea L.). The plants were induced aboveground or belowground with jasmonic acid (JA) and shoot feeding caterpillars (Pieris brassicae or P. rapae). We used Partial Least Squares—Discriminant Analysis (PLSDA) to integrate and visualize the relation between plant-emitted VOCs and the preference of female Cotesia glomerata. Overall, female wasps preferred JA-induced plants over controls, but they strongly preferred aboveground JA-induced plants over belowground JA-induced plants. PLSDA revealed that the emission of several monoterpenes was enhanced similarly in all JA-treated plants, whereas homoterpenes and sesquiterpenes increased exclusively in aboveground JA-induced plants. Wasps may use the ratio between these two classes of terpenes to discriminate between aboveground and belowground induced plants. Additionally, it shows that aboveground applied JA induces different VOC biosynthetic pathways than JA applied to the root. Our bioinformatic approach, thus, successfully identified which VOCs matched the preferences of the wasps in the various choice tests. Additionally, the analysis generated novel hypotheses about the role of JA as a signaling compound in aboveground and belowground induced responses in plants.
Aboveground-belowground interactions; Volatile organic compounds; Indirect defenses; Multivariate analysis; Terpenes; Jasmonic acid; Parasitoid preference; PLSDA
Many plant species show induced responses that protect them against exogenous attacks. These responses involve the production of many different bioactive compounds. Plant species belonging to the Brassicaceae family produce defensive glucosinolates, which may greatly influence their favorable nutritional properties for humans. Each responding compound may have its own dynamic profile and metabolic relationships with other compounds. The chemical background of the induced response is therefore highly complex and may therefore not reveal all the properties of the response in any single model.
This study therefore aims to describe the dynamics of the glucosinolate response, measured at three time points after induction in a feral Brassica, by a three-faceted approach, based on Principal Component Analysis. First the large-scale aspects of the response are described in a 'global model' and then each time-point in the experiment is individually described in 'local models' that focus on phenomena that occur at specific moments in time. Although each local model describes the variation among the plants at one time-point as well as possible, the response dynamics are lost. Therefore a novel method called the 'Crossfit' is described that links the local models of different time-points to each other.
Each element of the described analysis approach reveals different aspects of the response. The crossfit shows that smaller dynamic changes may occur in the response that are overlooked by global models, as illustrated by the analysis of a metabolic profiling dataset of the same samples.
Many induced responses in plants are systemic. Therefore, root-induced responses may alter leaf quality for shoot herbivores. Previously, we found that root and shoot application of jasmonic acid (JA) to feral Brassica oleracea both induced glucosinolates in the leaves. However, the types of glucosinolates that increased in root- and shoot induced plants were different. Here we analyse whether primary metabolites, such as sugars and amino acids, are also differentially affected. Moreover, we test whether chemical differences in root- and shoot-induced plants differentially affect growth of the generalist Mamestra brassicae and the specialist Pieris rapae. Comprehensive analysis of glucosinolates, amino acid and sugars with principal component analysis revealed that leaf chemical profiles were affected both by JA application and by the organ that was induced. Shoot-induction increased indole glucosinolates, whereas root-induction induced aliphatic glucosinolates in the leaves. Leaves of shoot-induced plants had lower total sugar and total amino acid levels, whereas in root-induced plants only total sugar levels were significantly decreased. (Iso)leucine responded significantly different from the general trend, which allowed us to discuss the potential role of Myb transcription factors which are coordinating JA-induced glucosinolate and amino acid responses in Arabidopsis thaliana. Both M. brassicae and P. rapae grew the slowest on leaves of shoot-induced plants. M. brassicae growth and survival was also reduced on root-induced plants, whereas P. rapae growth on these plants was similar to that on controls. Specialist and generalist herbivores thus are differentially affected by the chemical changes after root and shoot-JA application.
above-below ground interactions; amino acids; Brassica oleracea; glucosinolates; induced responses; jasmonic acid; Mamestra brassicae; Pieris rapae; sugars
Aliphatic glucosinolates are compounds which occur in high concentrations in Arabidopsis thaliana and other Brassicaceae species. They are important for the resistance of the plant to pest insects. Previously, the biosynthesis of these compounds was shown to be regulated by transcription factors MYB28 and MYB29. We now show that MYB28 and MYB29 are partially redundant, but in the absence of both, the synthesis of all aliphatic glucosinolates is blocked. Untargeted and targeted biochemical analyses of leaf metabolites showed that differences between single and double knock-out mutants and wild type plants were restricted to glucosinolates. Biosynthesis of long-chain aliphatic glucosinolates was blocked by the myb28 mutation, while short-chain aliphatic glucosinolates were reduced by about 50% in both the myb28 and the myb29 single mutants. Most remarkably, all aliphatic glucosinolates were completely absent in the double mutant. Expression of glucosinolate biosynthetic genes was slightly but significantly reduced by the single myb mutations, while the double mutation resulted in a drastic decrease in expression of these genes. Since the myb28myb29 double mutant is the first Arabidopsis genotype without any aliphatic glucosinolates, we used it to establish the relevance of aliphatic glucosinolate biosynthesis to herbivory by larvae of the lepidopteran insect Mamestra brassicae. Plant damage correlated inversely to the levels of aliphatic glucosinolates observed in those plants: Larval weight gain was 2.6 fold higher on the double myb28myb29 mutant completely lacking aliphatic glucosinolates and 1.8 higher on the single mutants with intermediate levels of aliphatic glucosinolates compared to wild type plants.
The composition of secondary metabolites and the nutritional value of a plant both determine herbivore preference and performance. The genetically determined glucosinolate pattern of Barbarea vulgaris can be dominated by either glucobarbarin (BAR-type) or by gluconasturtiin (NAS-type). Because of the structural differences, these glucosinolates may have different effects on herbivores. We compared the two Barbarea chemotypes with regards to the preference and performance of two lepidopteran herbivores, using Mamestra brassicae as a generalist and Pieris rapae as a specialist. The generalist and specialist herbivores did not prefer either chemotype for oviposition. However, larvae of the generalist M. brassicae preferred to feed and performed best on NAS-type plants. On NAS-type plants, 100% of the M. brassicae larvae survived while growing exponentially, whereas on BAR-type plants, M. brassicae larvae showed little growth and a mortality of 37.5%. In contrast to M. brassicae, the larval preference and performance of the specialist P. rapae was unaffected by plant chemotype. Total levels of glucosinolates, water soluble sugars, and amino acids of B. vulgaris could not explain the poor preference and performance of M. brassicae on BAR-type plants. Our results suggest that difference in glucosinolate chemical structure is responsible for the differential effects of the B. vulgaris chemotypes on the generalist herbivore.
Electronic supplementary material
The online version of this article (doi:10.1007/s10886-007-9424-9) contains supplementary material, which is available to authorized users.
Glucosinolates; Oviposition; Performance; Polymorphism; Barbarea vulgaris; Pieris rapae; Mamestra brassicae; Co-evolution
Through artificial selection, domesticated plants often contain modified levels of primary and secondary metabolites compared to their wild progenitors. It is hypothesized that the changed chemistry of cultivated plants will affect the performance of insects associated with these plants. In this paper, the development of several specialist and generalist herbivores and their endoparasitoids were compared when reared on a wild and cultivated population of cabbage, Brassica oleracea, and a recently established feral Brassica species. Irrespective of insect species or the degree of dietary specialization, herbivores and parasitoids developed most poorly on the wild population. For the specialists, plant population influenced only development time and adult body mass, whereas for the generalists, plant populations also affected egg-to-adult survival. Two parasitoid species, a generalist (Diadegma fenestrale) and a specialist (D. semiclausum), were reared from the same host (Plutella xylostella). Performance of D. semiclausum was closely linked to that of its host, whereas the correlation between survival of D. fenestrale and host performance was less clear. Plants in the Brassicaceae characteristically produce defense-related glucosinolates (GS). Levels of GS in leaves of undamaged plants were significantly higher in plants from the wild population than from the domesticated populations. Moreover, total GS concentrations increased significantly in wild plants after herbivory, but not in domesticated or feral plants. The results of this study reveal that a cabbage cultivar and plants from a wild cabbage population exhibit significant differences in quality in terms of their effects on the growth and development of insect herbivores and their natural enemies. Although cultivated plants have proved to be model systems in agroecology, we argue that some caution should be applied to evolutionary explanations derived from studies on domesticated plants, unless some knowledge exists on the history of the system under investigation.
Crucifer; Diadegma fenestrale; Diadegma semiclausum; Direct defense; Glucosinolates; Induction; Mamestra brassicae; Pieris rapae; Plutella xylostella
Jasmonic acid (JA) is a key hormone involved in plant defense responses. The effect of JA treatment of cabbage plants on their acceptability for oviposition by two species of cabbage white butterflies, Pieris rapae and P. brassicae, was investigated. Both butterfly species laid fewer eggs on leaves of JA-treated plants compared to control plants. We show that this is due to processes in the plant after JA treatment rather than an effect of JA itself. The oviposition preference for control plants is adaptive, as development time from larval hatch until pupation of P. rapae caterpillars was longer on JA-treated plants. Total glucosinolate content in leaf surface extracts was similar for control and treated plants; however, two of the five glucosinolates were present in lower amounts in leaf surface extracts of JA-treated plants. When the butterflies were offered a choice between the purified glucosinolate fraction isolated from leaf surface extracts of JA-treated plants and that from control plants, they did not discriminate. Changes in leaf surface glucosinolate profile, therefore, do not seem to explain the change in oviposition preference of the butterflies after JA treatment, suggesting that as yet unknown infochemicals are involved.
Host plant selection; Glucosinolates; Pieris rapae; Pieris brassicae; Cabbage; Performance; Octadecanoid pathway
Plants respond to herbivore attack by rapidly inducing defenses that are mainly regulated by jasmonic acid (JA). Due to the systemic nature of induced defenses, attack by root herbivores can also result in a shoot response and vice versa, causing interactions between above- and belowground herbivores. However, little is known about the molecular mechanisms underlying these interactions. We investigated whether plants respond differently when roots or shoots are induced. We mimicked herbivore attack by applying JA to the roots or shoots of Brassica oleracea and analyzed molecular and chemical responses in both organs. In shoots, an immediate and massive change in primary and secondary metabolism was observed. In roots, the JA-induced response was less extensive and qualitatively different from that in the shoots. Strikingly, in both roots and shoots we also observed differential responses in primary metabolism, development as well as defense specific traits depending on whether the JA induction had been below- or aboveground. We conclude that the JA response is not only tissue-specific but also dependent on the organ that was induced. Already very early in the JA signaling pathway the differential response was observed. This indicates that both organs have a different JA signaling cascade, and that the signal eliciting systemic responses contains information about the site of induction, thus providing plants with a mechanism to tailor their responses specifically to the organ that is damaged.