Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.)

1.  Plants are simultaneously attacked by multiple herbivores and pathogens. While some plant defences act synergistically, others trade-off against each other. Such trade-offs among resistances to herbivores and pathogens are usually explained by the costs of resistance, i.e. resource limitations compromising a plant's overall defence.
2.  Here, we demonstrate that trade-offs can also result from direct negative interactions among defensive traits. We studied cyanogenesis (release of HCN) of lima bean (Fabaceae: Phaseolus lunatus ) and effects of this efficient anti-herbivore defence on resistance to a fungal pathogen (Melanconiaceae: Colletotrichum gloeosporioides ).
3.  Leaf tissue destruction by fungal growth was significantly higher on high cyanogenic (HC) lima bean accessions than on low cyanogenic (LC) plants. The susceptibility of HC accessions to the fungal pathogen was strongly correlated to reduced activity of resistance-associated polyphenol oxidases (PPOs) in leaves of these plants. LC accessions, in contrast, showed high PPO activity, which was correlated with distinct resistance to C. gloeosporioides .
4.  Experimentally applied, gaseous HCN reduced PPO activity and significantly increased the size of lesions caused by C. gloeosporioides in LC leaves.
5.  Field observations of a wild lima bean population in Mexico revealed a higher infection rate of HC compared to LC plant individuals. The types of lesions observed on the different cyanogenic plants in nature were similar to those observed on HC and LC plants in the laboratory.
6.   Synthesis. We suggest that cyanogenesis of lima bean directly trades off with plant defence against fungal pathogens and that the causal mechanism is the inhibition of PPOs by HCN. Our findings provide a functional explanation for the observed phenomenon of the low resistance of HC lima beans in nature.     

Comparing responses of generalist and specialist herbivores to various cyanogenic plant features

Plants are obliged to defend themselves against multiple generalist and specialist herbivores. Whereas plant cyanogenesis is considered an efficient defence against generalists, it is thought to affect specialists less. In the present study, we analysed the function of various cyanogenic features of lima bean [Phaseolus lunatus L. (Fabaceae)] during interaction with different herbivores. Three cyanogenic features were analysed, i.e., cyanogenic potential (HCNp; concentration of cyanogenic precursors), β‐glucosidase activity, and cyanogenic capacity (HCNc; release of cyanide per unit time). In no‐choice and free‐choice feeding trials, five lima bean accessions were offered to generalist desert locust [Schistocerca gregaria Forskål (Orthoptera: Acrididae)] and specialist Mexican bean beetle [Epilachna varivestis Mulsant (Coleoptera: Coccinellidae)]. The HCNc was the most important parameter determining host plant selection by generalists, whereas choice behaviour of specialists was strongly affected by HCNp. Although locusts were effectively repelled by high HCNc, this cue was misleading for the detection of suitable host plants, as extensive consumption of low HCNc plant material resulted in strong intoxication of locusts. Balancing cyanide in consumed leaf area, the quantitative release of gaseous cyanide during feeding, and cyanide in faeces suggested that specialists metabolized significantly lower rates of cyanide per consumed leaf material than generalists. We hypothesize that specialists are able to avoid toxic concentrations of cyanide by using HCNp rather than HCNc as a cue for host plant quality, and that they exhibit mechanisms that reduce incorporation of host plant cyanide.     

CO2‐mediated changes of plant traits and their effects on herbivores are determined by leaf age

1. Concentration of atmospheric CO₂ is predicted to double during the 21st century. However, quantitative effects of increased CO₂ levels on natural herbivore–plant interactions are still little understood. 2. In this study, we assess whether increased CO₂ quantitatively affects multiple defensive and nutritive traits in different leaf stages of cyanogenic wildtype lima bean plants (Phaseolus lunatus), and whether plant responses influence performance and choice behaviour of a natural insect herbivore, the Mexican bean beetle (Epilachna varivestis). 3. We cultivated lima bean plants in climate chambers at ambient, 500, 700, and 1000 ppm CO₂ and analysed cyanogenic precursor concentration (nitrogen‐based defence), total phenolics (carbon‐based defence), leaf mass per area (LMA; physical defence), and soluble proteins (nutritive parameter) of three defined leaf age groups. 4. In young leaves, cyanide concentration was the only parameter that quantitatively decreased in response to CO₂ treatments. In intermediate and mature leaves, cyanide and protein concentrations decreased while total phenolics and LMA increased. 5. Depending on leaf stage, CO₂‐mediated changes in leaf traits significantly affected larval performance and choice behaviour of adult beetles. We observed a complete shift from highest herbivore damage in mature leaves under natural CO₂ to highest damage of young leaves under elevated CO_2. Our study shows that leaf stage is an essential factor when considering CO₂‐mediated changes of plant defences against herbivores. Since in the long run preferred consumption of young leaves can strongly affect plant fitness, variable effects of elevated CO₂ on different leaf stages should receive highlighted attention in future research.     

Differential effects of type and quantity of leaf damage on growth,reproduction and defence of lima bean (Phaseolus lunatus L.)

Folivores are major plant antagonists in most terrestrial ecosystems. However, the quantitative effects of leaf area loss on multiple interacting plant traits are still little understood. We sought to contribute to filling this lack of understanding by applying different types of leaf area removal (complete leaflets versus leaflet parts) and degrees of leaf damage (0, 33 and 66%) to lima bean (Phaseolus lunatus) plants. We quantified various growth and fitness parameters including above‐ and belowground biomass as well as the production of reproductive structures (fruits, seeds). In addition, we measured plant cyanogenic potential (HCNp; direct chemical defence) and production of extrafloral nectar (EFN; indirect defence). Leaf damage reduced above‐ and belowground biomass production in general, but neither variation in quantity nor type of damage resulted in different biomass. Similarly, the number of fruits and seeds was significantly reduced in all damaged plants without significant differences between treatment groups. Seed mass, however, was affected by both type and quantity of leaf damage. Leaf area loss had no impact on HCNp, whereas production of EFN decreased with increasing damage. While EFN production was quantitatively affected by leaf area removal, the type of damage had no effect. Our study provides a thorough analysis of the quantitative and qualitative effects of defoliation on multiple productivity‐related and defensive plant traits and shows strong differences in plant response depending on trait. Quantifying such plant responses is vital to our understanding of the impact of herbivory on plant fitness and productivity in natural and agricultural ecosystems.     

Do plants use airborne cues to recognize herbivores on their neighbours?

Plants show defensive responses after exposure to volatiles from neighbouring plants infested by herbivores. When a plant’s neighbours host only species of herbivores that do not feed on the plant itself, the plant can conserve energy by maintaining a low defence level. An intriguing question is whether plants respond differently to volatiles from plants infested by herbivores that pose greater or lesser degrees of danger. We examined the secretion of extrafloral nectar (EFN) in lima bean plants exposed to volatiles from cabbage plants infested by common cutworm, two-spotted spider mites, or diamondback moth larvae. Although the first two herbivore species feed on lima bean plants, diamondback moth larvae do not. As a control, lima bean plants were exposed to volatiles from uninfested cabbage plants. Only when exposed to volatiles from cabbage plants infested by spider mites did lima bean plants significantly increase their EFN secretion compared with the control. Increased EFN secretion can function as an indirect defence by supplying the natural enemies of herbivores with an alternative food source. Of the three herbivore species, spider mites were the most likely to move from cabbage plants to lima bean plants and presumably posed the greatest threat. Although chemical analyses showed differences among treatments in volatiles produced by herbivore-infested cabbage plants, which compounds or blends triggered the increased secretion of EFN by lima bean plants remains unclear. Thus, our results show that plants may tune their defence levels according to herbivore risk level.     

Chemical defense lowers plant competitiveness

Both plant competition and plant defense affect biodiversity and food web dynamics and are central themes in ecology research. The evolutionary pressures determining plant allocation toward defense or competition are not well understood. According to the growth–differentiation balance hypothesis (GDB), the relative importance of herbivory and competition have led to the evolution of plant allocation patterns, with herbivore pressure leading to increased differentiated tissues (defensive traits), and competition pressure leading to resource investment towards cellular division and elongation (growth-related traits). Here, we tested the GDB hypothesis by assessing the competitive response of lima bean (Phaseolus lunatus) plants with quantitatively different levels of cyanogenesis—a constitutive direct, nitrogen-based defense against herbivores. We used high (HC) and low cyanogenic (LC) genotypes in different competition treatments (intra-genotypic, inter-genotypic, interspecific), and in the presence or absence of insect herbivores (Mexican bean beetle, Epilachna varivestis) to quantify vegetative and generative plant parameters (above and belowground biomass as well as seed production). Highly defended HC-plants had significantly lower aboveground biomass and seed production than LC-plants when grown in the absence of herbivores implying significant intrinsic costs of plant cyanogenesis. However, the reduced performance of HC- compared to LC-plants was mitigated in the presence of herbivores. The two plant genotypes exhibited fundamentally different responses to various stresses (competition, herbivory). Our study supports the GDB hypothesis by demonstrating that competition and herbivory affect different plant genotypes differentially and contributes to understanding the causes of variation in defense within a single plant species.     

Ecological trade-offs between jasmonic acid-dependent direct and indirect plant defences in tritrophic interactions

Recent studies on plants genetically modified in jasmonic acid (JA) signalling support the hypothesis that the jasmonate family of oxylipins plays an important role in mediating direct and indirect plant defences. However, the interaction of two modes of defence in tritrophic systems is largely unknown. In this study, we examined the preference and performance of a herbivorous leafminer (Liriomyza huidobrensis) and its parasitic wasp (Opius dissitus) on three tomato genotypes: a wild-type (WT) plant, a JA biosynthesis (spr2) mutant, and a JA-overexpression 35S::prosys plant. Their proteinase inhibitor production and volatile emission were used as direct and indirect defence factors to evaluate the responses of leafminers and parasitoids. Here, we show that although spr2 mutant plants are compromised in direct defence against the larval leafminers and in attracting parasitoids, they are less attractive to adult flies compared with WT plants. Moreover, in comparison to other genotypes, the 35S::prosys plant displays greater direct and constitutive indirect defences, but reduced success of parasitism by parasitoids. Taken together, these results suggest that there are distinguished ecological trade-offs between JA-dependent direct and indirect defences in genetically modified plants whose fitness should be assessed in tritrophic systems and under natural conditions.     

Short signalling distances make plant communication a soliloquy

Plants respond to attack by herbivores or pathogens with the release of volatile organic compounds. Neighbouring plants can receive these volatiles and consecutively induce their own defence arsenal. This ‘plant communication’, however, appears counterintuitive when it benefits independent and genetically unrelated receivers, which may compete with the emitter. As a solution to this problem, a role for volatile compounds in within-plant signalling has been predicted. We used wild-type lima bean (Phaseolus lunatus) to quantify under field conditions the distances over which volatile signals move, and thereby determine whether these cues will mainly trigger resistance in other parts of the same plant or in independent plants. Independent receiver plants exhibited airborne resistance to herbivores or pathogens at maximum distances of 50 cm from a resistance-expressing emitter. In undisturbed clusters of lima bean, over 80 per cent of all leaves that were located around a single leaf at this distance were other leaves of the same plant, whereas this percentage dropped below 50 per cent at larger distances. Under natural conditions, resistance-inducing volatiles of lima bean move over distances at which most leaves that can receive the signal still belong to the same plant.     

Attraction of Phytoseiulus persimilis (Acari: Phytoseiidae) towards volatiles from various Tetranychus urticae-infested plant species

Plants infested with the spider mite Tetranychus urticae Koch, may indirectly defend themselves by releasing volatiles that attract the predatory mite Phytoseiulus persimilis Athias-Henriot. Several plants from different plant families that varied in the level of spider mite acceptance were tested in an olfactometer. The predatory mites were significantly attracted to the spider mite-infested leaves of all test plant species. No differences in attractiveness of the infested plant leaves were found for predatory mites reared on spider mites on the different test plants or on lima bean. Thus, experience with the spider mite-induced plant volatiles did not affect the predatory mites. Jasmonic acid was applied to ginkgo leaves to induce a mimic of a spider mite-induced volatile blend, because the spider mites did not survive when incubated on ginkgo. The volatile blend induced in ginkgo by jasmonic acid was slightly attractive to predatory mites. Plants with a high degree of direct defence were thought to invest less in indirect defence than plants with a low degree of direct defence. However, plants that had a strong direct defence such as ginkgo and sweet pepper, did emit induced volatiles that attracted the predatory mite. This indicates that a combination of direct and indirect defence is to some extent compatible in plant species.     

Plant stages with biotic,indirect defences are more palatable and suffer less herbivory than their undefended counterparts

Plants have evolved several anti‐herbivory strategies, including direct defences, such as mechanical and chemical defences, and indirect or biotic defences, such as the recruitment of defending animals. We examined whether the investment plants make in direct defences differs between those which do and do not invest in biotic defences, by comparing standing herbivory and palatability of congeneric species with and without indirect defences at two ontogenetic stages: before and after the onset of indirect defences. We used Cordia alliodora and Croton suberosus as the species with indirect defences and Cordia elaeagnoides and Croton pseudoniveus as the species without indirect defences. We predicted that herbivores would prefer to eat species and stages with indirect defences to those without them. As predicted, we found that herbivores preferred species and ontogenetic stages with indirect defences in all cases. Overall, however, natural levels of herbivory were lower in species with indirect defences. We conclude that indirect defences offer effective protection against herbivores and posit that their recruitment allows plants to reduce investment in other defence mechanisms. Our results support the notion that plants trade‐off between direct and indirect defensive strategies. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 536–543.     

Jasmonate-deficient plants have reduced direct and indirect defences against herbivores

Plants employ a variety of defence mechanisms, some of which act directly by having a negative effect on herbivores and others that act indirectly by attracting natural enemies of herbivores. In this study we asked if a common jasmonate‐signalling pathway links the regulation of direct and indirect defences in plants. We examined the performance of herbivores (direct defence) and the attraction of natural enemies of herbivores (indirect defence) to wild‐type tomato plants and mutant plants that are deficient in the production of the signalling hormone jasmonic acid. Wild‐type plants supported lower survivorship of caterpillars compared with jasmonic acid‐deficient plants. Damaged wild‐type plants were more attractive to predaceous mites compared with undamaged wild‐type plants, whereas damaged jasmonate‐deficient plants were not more attractive to predators. Damaged wild‐type plants induced a greater production of volatile compounds (primarily the sesquiterpene β‐caryophyllene and the monoterpenes α‐pinene, β‐pinene, 2‐carene and β‐phellandrene) compared with damaged jasmonate‐deficient plants. Treating jasmonate‐deficient plants with exogenous jasmonic acid restored both the direct and indirect defence capabilities, demonstrating that jasmonic acid is an essential regulatory component for the expression of direct and indirect plant defence.     

Analyzing plant defenses in nature

A broad range of chemical plant defenses against herbivores has been studied extensively under laboratory conditions. In many of these cases there is still little understanding of their relevance in nature. In natural systems, functional analyses of plant traits are often complicated by an extreme variability, which affects the interaction with higher trophic levels. Successful analyses require consideration of the numerous sources of variation that potentially affect the plant trait of interest. In our recent study on wild lima bean (Phaseolus lunatus L.) in South Mexico, we applied an integrative approach combining analyses for quantitative correlations of cyanogenic potential (HCNp; the maximum amount of cyanide that can be released from a given tissue) and herbivory in the field with subsequent feeding trials under controlled conditions. This approach allowed us to causally explain the consequences of quantitative variation of HCNp on herbivore-plant interactions in nature and highlights the importance of combining data obtained in natural systems with analyses under controlled conditions.Key words: natural systems, plant defensive traits, optimal defense hypothesis (ODH), cyanogenesis, lima bean, Phaseolus lunatus L., plant-herbivore interaction, plant-pathogen interaction, multiple defense syndromesAnalyzing plant defenses against herbivores in nature is often complicated by an extreme variability in multiple factors. Plant populations generally show high genetic variability resulting in substantial intraspecific variation of plant traits.¹ In addition to genotypic variability, phenotypic plasticity of plants is a source of variation.² At the level of individual plants, expression of defensive traits strongly depends on plant organ and ontogeny of plants or plant parts. Within an individual plant, it is quite common for reproductive structures and young leaves to be better chemically defended than older leaf tissues. To explain these within-plant variations of defenses, the optimal defense hypothesis (ODH) was formulated. Concerning the variability of chemical defenses of leaves, the ODH predicts that within the total foliage of a plant, young leaves make a larger contribution to plant fitness than old leaves as they have a higher potential photosynthetic value resulting from a longer expected life-time.^3–5 In addition, younger leaves are often more nutritious and thus more attractive to herbivores⁶ and should consequently be better defended.⁷ In this line, the basic assumption of the ODH is that three main factors—cost of defense, risk of attack and value of the respective plant organ—determine the investment in defensive secondary metabolites.^8,9 Thus, the higher the risk of a given plant tissue to be consumed by herbivores and the higher its value for plant fitness, the more energy should be allocated to its defense.^10,11 Beyond genotypic and ontogenetic variability of a given defense, potential co-variation with other defensive or nutritive traits expressed by the same plant individual can strongly affect its efficiency as defense against herbivores.^12,13 In addition to these endogenous sources of defensive variability, the expression of plant traits strongly depends on multiple external factors such as temperature or availability of plant nutrients, water or light (Fig. 1).¹⁴ At the same time, the outcome of herbivore-plant interactions is crucially determined by biotic interactions. Plant interactions with mutualistic microorganisms such as Rhizobia, mycorrhiza and above-ground fungal endophytes as well as tri-trophic interactions with predators and parasitoids of herbivores can all strongly impact plant fitness.¹⁵Open in a separate windowFigure 1Factors influencing variability of plant defenses. Plant defensive traits are affected by various endogenous and external factors. Endogenous factors comprise plant genotype and ontogeny of plants or plant parts. External factors can be categorized as abiotic or biotic. Important abiotic factors that can influence plant defenses are light exposure, temperature, soil salinity, as well as water and nutrient availability. Biotic factors that can have an effect on plant defense are interspecific interaction with Rhizobia (in the case of legumes), mycorrhizal and endophytic fungi, pathogens as well as the interaction with conspecifics or different plant species.Variability in herbivore-plant interactions can also be associated with herbivore variation. Different attackers of a particular plant species might be affected in different ways by toxins in food plants (Fig. 1). The efficiency of a specific defensive compound can also depend on the feeding mode, i.e., sucking or chewing, as well as on the degree of specialization of the herbivore to the respective plant.¹⁶ Defenses mediated by secondary plant compounds are generally believed not to affect specialist herbivores, because of their capacity to tolerate or to detoxify defensive compounds of their hosts by behavioral or physiological adaptations.^17–20 In this context, the specialist herbivore paradigm predicts that adapted herbivores are less affected by a given chemical defense than generalists,^21,22 although exceptions have been noted.^23–25While it is important to consider these numerous sources of variation affecting the outcome of herbivore-plant interactions when designing functional studies, a significant fraction of the variability in natural systems will always remain unidentified. Consequently, approaches combining field observations with experiments under controlled conditions provide a powerful tool to uncover functional interactions between plants and their multiple antagonists in nature.In a recent study, we analyzed the importance of wild lima bean''s cyanogenesis—i.e., the release of toxic hydrogen cyanide from preformed precursors in response to cell damage—as plant defense at a natural site in South Mexico.²⁵ Although cyanogenesis is generally considered an efficient direct defense against herbivores, in numerous studies plant cyanide production had little or no effect on herbivores.^26–28 One would like to think that most of these inconsistencies in cyanogenesis-based herbivore defense efficiency could be explained by one or more sources of variation mentioned above. Nevertheless, field studies analyzing the action of plant cyanogenesis on a quantitative basis have been scarce. In our study, a two-step approach was used to gain insight into the function of cyanogenesis in nature.²⁵ First, cyanide concentration and herbivore damage were quantified by measuring removed leaf area of individual leaves derived from different individual plants while considering microclimate conditions. Significant negative correlations between cyanogenesis and leaf damage were observed. Second, since existing correlations do not necessarily indicate causal associations, we conducted consecutive feeding experiments under controlled conditions. To consider natural variability of lima beans'' cyanogenesis observed in nature in our analysis, we prepared clones from field-grown plants with different but defined cyanogenic features. These clonal plants showed high constancy of cyanogenic traits compared to their respective mother plants and thus, could be used in comparative analyses. Every effort was made to duplicate natural conditions and so herbivore species selected for feeding trials represented those identified in the field as the most important plant consumers at the respective site (pers. observ.). Feeding trials supported our hypothesis that cyanogenesis has quantitative effects on herbivore behavior in nature and explained the negative correlation of lima bean''s cyanogenesis and herbivory observed in the field.Analytical approaches combining field observations with controlled experiments help to explain natural patterns and may represent a powerful methodological approach for functional analyses of herbivore-plant interactions.     

Aridity shapes cyanogenesis cline evolution in white clover (Trifolium repens L.)

Adaptive differentiation between populations is often proposed to be the product of multiple interacting selective pressures, although empirical support for this is scarce. In white clover, populations show adaptive differentiation in frequencies of cyanogenesis, the ability to produce hydrogen cyanide after tissue damage. This polymorphism arises through independently segregating polymorphisms for the presence/absence of two required cyanogenic components, cyanogenic glucosides and their hydrolysing enzyme. White clover populations worldwide have evolved a series of recurrent, climate‐associated clines, with higher frequencies of cyanogenic plants in warmer locations. These clines have traditionally been hypothesized to reflect a fitness trade‐off between chemical defence in herbivore‐rich areas (warmer climates) and energetic costs of producing cyanogenic components in areas of low herbivore pressure (cooler climates). Recent observational studies suggest that cyanogenic components may also be beneficial in water‐stressed environments. We investigated fitness trade‐offs associated with temperature‐induced water stress in the cyanogenesis system using manipulative experiments in growth chambers and population surveys across a longitudinal precipitation gradient in the central United States. We find that plants producing cyanogenic glucosides have higher relative fitness in treatments simulating a moderate, persistent drought stress. In water‐neutral treatments, there are energetic costs to producing cyanogenic components, but only in treatments with nutrient stress. These fitness trade‐offs are consistent with cyanogenesis frequencies in natural populations, where we find clinal variation in the proportion of plants producing cyanogenic glucosides along the precipitation gradient. These results suggest that multiple selective pressures interact to maintain this adaptive polymorphism and that modelling adaptation will require knowledge of environment‐specific fitness effects.     

Qualitative variability of lima bean's VOC bouquets and its putative ecological consequences

Studies on direct and indirect defenses of lima bean (Phaseolus lunatus L.) revealed a quantitative trade-off between cyanogenesis and the total quantitative release of herbivore-induced volatile organic compounds (VOCs). In this addendum we focus on the qualitative variability in the VOC bouquets. We found intraspecific and ontogenetic variation. Five out of eleven lima bean accessions lacked particular VOCs in the bouquets released from secondary and/or primary leaves. These compounds (cis-3-hexenyl acetate, methyl salicylate and methyl jasmonate) can induce and prime indirect defenses in neighboring plants. Thus, the variability in VOC quality as described here might have substantial effects on plant-plant communication.Key words: indirect defense, herbivore-induced plant volatiles, Phaseolus lunatus, trade-off, tritrophic interactions, plant-plant communicationPlants can be attacked by multiple enemies and accordingly have evolved multiple defense strategies comprising direct and indirect mechanisms. Lima bean (Fabaceae: Phaseolus lunatus L.) represents a well established experimental plant for the analysis of three defenses: herbivore-induced volatile organic compounds (VOCs), extrafloral nectar (EFN) and plant cyanogenesis. Herbivore-induced VOCs have manifold functions associated to indirect plant defenses. For example, VOCs attract arthropod predators or parasitoids and thus can act as an indirect defense.^1–5 They also may be perceived by neighboring, yet-undamaged plant individuals (plant-plant signaling) or plant parts (within-plant signaling) and they prime or induce defensive responses.⁶ VOC-exposed plants may upregulate the secretion of EFN^7–9 or the release of VOCs.¹⁰ In addition to these indirect defenses, lima bean shows cyanogenesis as a direct defense. Cyanogenesis means the release of toxic hydrogen cyanide (HCN) from preformed precursors in response to cell damage¹¹ and is considered a constitutive direct defense against herbivores¹² (but see ref. ¹³).Recently we demonstrated that cyanogenesis and total release of VOC in lima bean are negatively correlated to each other.¹⁴ Accessions characterized by strong cyanogenesis in secondary leaves released little amounts of VOCs in response to jasmonic acid (JA) treatment, whereas total VOC production in accessions with low cyanide concentrations was high. Interestingly, these findings also held true on the ontogenetic level, since primary leaves generally showed low concentrations of cyanide and released high amounts of VOCs. However, the question remained unanswered whether these differences are merely of quantitative or also of qualitative nature. We therefore selected eleven accessions from the larger set of lima beans that had been used in our previous study and searched for qualitative differences in their VOC bouquets.Eight out of the eleven volatiles that were quantified in our study were consistently released from both primary and secondary leaves of all accessions. However, five accessions indeed lacked individual compounds in secondary or primary leaves. For example, we did not detect cis-3-hexenyl acetate in the headspace of secondary leaves of accessions CV_2357 and CV_8078. The latter accession also lacked methyl salicylate in all individual plants analyzed (N = 10 plants). In contrast to secondary leaves, primary leaves of these accessions showed the complete blend of eleven VOCs. While CV_2357 and CV_8078 showed qualitative variability in VOC blends depending on leaf developmental stage, the accessions WT_2233 and WT_PYU consistently lacked methyl jasmonate in both, secondary and primary leaves. Strikingly, accession CV_8073, which was characterized by high quantitative release of cis-3-hexenyl acetate and methyl salicylate from secondary and primary leaves, showed a very low release of methyl jasmonate from secondary leaves—and lacked this compound completely in the headspace of primary leaves (Fig. 1).Open in a separate windowFigure 1Qualitative variation in VOC mixtures of lima bean. Secondary (A) and primary leaves (B) of cultivated (CV) and wildtype (WT) lima beans characterized by high (HC) or low (LC) concentration of cyanogenic precursors in secondary leaves were analysed for release of three selected VOC compounds. Volatiles were collected in a closed-loop stripping over an experimental period of exactly 24 hrs. Values shown represent the mean (±SE) of five plants per accession. Different letters on top of the columns indicate means that differ significantly (according LSD post-hoc analysis after one-way ANOVA. Statistical analyses were conducted separately for each compound and each leaf stage).In addition to qualitative differences, the quantitative release of volatiles was significantly different among the accessions. This holds true for the three compounds we have focused on in this addendum (Fig. 1) as well as for all eleven volatiles that we have quantified in our previous study (data not shown). However, while quantitative release of total VOCs from secondary leaves was negatively correlated to their cyanide concentration,¹⁴ the qualitative differences in VOC composition were not that strictly correlated to cyanogenesis. Accessions CV_2357, CV_8078, WT_2233 and WT_PYU were characterized by high cyanogenic secondary leaves—and lacked at least one compound. However, also the generally low cyanogenic primary leaves did not always show the complete VOC blend (Fig. 1B). These findings demonstrate a high qualitative variability of VOC composition that did depend neither on cyanogenic leaf features nor on leaf ontogeny.So far it remains elusive whether the observed intraspecific and ontogenetic variability is of relevance in natural systems, but ecological effects are highly likely. For some tritrophic systems an intriguing degree of sophistication in the communication between plants and the third trophic level has been demonstrated: bouquet compositions can provide specific information on the identity of the attacking herbivore and, hence, on its suitability for the prey-seeking carnivores.¹⁵ Variability in the composition of VOC compositions as observed for lima bean (and as also known for corn, cotton or cabbage^16–20) may compromise the reliability of herbivore-specific signals across a range of plant genotypes,²¹ although the ability of parasitoids to learn and associate successful foraging and egg-laying experience with the encountered odor pattern may help them in part overcome this problem.^22,23In contrast to tritrophic interactions, the efficiency of volatiles in plant-plant signaling appears more restricted to specific compounds. Recent studies on lima bean demonstrated that cis-3-hexenyl acetate causes priming or induction of extrafloral nectar.^7,24 In other plant species, the release of gaseous methyl jasmonate in response to herbivore attack has been demonstrated to induce the synthesis of proteinase-inhibitors, which represent an efficient defense against herbivores.^6,25 Methyl salicylate has been reported to be an important elicitor of resistance responses directed towards pathogens and herbivores^26,27 and as a carnivore-attractant.^28,29 Thus, the quantitative variation or a complete lacking of single biological active compounds of volatile blends may have dramatic effects on ecological interactions. We suggest that different defense strategies may be realized in these lima bean accessions: some genotypes have evolved strong cyanogenesis in secondary leaves and now ‘pay’ for this efficient direct defense with reduced or lost abilities for indirect defense and/or plant-plant communication, while others invest less in cyanogenesis and are more “communicative” concerning the third trophic level and conspecifics.     

Herbivores and plant defences affect selection on plant reproductive traits more strongly than pollinators

Pollinators and herbivores can both affect the evolutionary diversification of plant reproductive traits. However, plant defences frequently alter antagonistic and mutualistic interactions, and therefore, variation in plant defences may alter patterns of herbivore‐ and pollinator‐mediated selection on plant traits. We tested this hypothesis by conducting a common garden field experiment using 50 clonal genotypes of white clover (Trifolium repens) that varied in a Mendelian‐inherited chemical antiherbivore defence—the production of hydrogen cyanide (HCN). To evaluate whether plant defences alter herbivore‐ and/or pollinator‐mediated selection, we factorially crossed chemical defence (25 cyanogenic and 25 acyanogenic genotypes), herbivore damage (herbivore suppression) and pollination (hand pollination). We found that herbivores weakened selection for increased inflorescence production, suggesting that large displays are costly in the presence of herbivores. In addition, herbivores weakened selection on flower size but only among acyanogenic plants, suggesting that plant defences reduce the strength of herbivore‐mediated selection. Pollinators did not independently affect selection on any trait, although pollinators weakened selection for later flowering among cyanogenic plants. Overall, cyanogenic plant defences consistently increased the strength of positive directional selection on reproductive traits. Herbivores and pollinators both strengthened and weakened the strength of selection on reproductive traits, although herbivores imposed ~2.7× stronger selection than pollinators across all traits. Contrary to the view that pollinators are the most important agents of selection on reproductive traits, our data show that selection on reproductive traits is driven primarily by variation in herbivory and plant defences in this system.     

Mechanisms and ecological consequences of plant defence induction and suppression in herbivore communities

Background Plants are hotbeds for parasites such as arthropod herbivores, which acquire nutrients and energy from their hosts in order to grow and reproduce. Hence plants are selected to evolve resistance, which in turn selects for herbivores that can cope with this resistance. To preserve their fitness when attacked by herbivores, plants can employ complex strategies that include reallocation of resources and the production of defensive metabolites and structures. Plant defences can be either prefabricated or be produced only upon attack. Those that are ready-made are referred to as constitutive defences. Some constitutive defences are operational at any time while others require activation. Defences produced only when herbivores are present are referred to as induced defences. These can be established via de novo biosynthesis of defensive substances or via modifications of prefabricated substances and consequently these are active only when needed. Inducibility of defence may serve to save energy and to prevent self-intoxication but also implies that there is a delay in these defences becoming operational. Induced defences can be characterized by alterations in plant morphology and molecular chemistry and are associated with a decrease in herbivore performance. These alterations are set in motion by signals generated by herbivores. Finally, a subset of induced metabolites are released into the air as volatiles and function as a beacon for foraging natural enemies searching for prey, and this is referred to as induced indirect defence.Scope The objective of this review is to evaluate (1) which strategies plants have evolved to cope with herbivores and (2) which traits herbivores have evolved that enable them to counter these defences. The primary focus is on the induction and suppression of plant defences and the review outlines how the palette of traits that determine induction/suppression of, and resistance/susceptibility of herbivores to, plant defences can give rise to exploitative competition and facilitation within ecological communities “inhabiting” a plant.Conclusions Herbivores have evolved diverse strategies, which are not mutually exclusive, to decrease the negative effects of plant defences in order to maximize the conversion of plant material into offspring. Numerous adaptations have been found in herbivores, enabling them to dismantle or bypass defensive barriers, to avoid tissues with relatively high levels of defensive chemicals or to metabolize these chemicals once ingested. In addition, some herbivores interfere with the onset or completion of induced plant defences, resulting in the plant’s resistance being partly or fully suppressed. The ability to suppress induced plant defences appears to occur across plant parasites from different kingdoms, including herbivorous arthropods, and there is remarkable diversity in suppression mechanisms. Suppression may strongly affect the structure of the food web, because the ability to suppress the activation of defences of a communal host may facilitate competitors, whereas the ability of a herbivore to cope with activated plant defences will not. Further characterization of the mechanisms and traits that give rise to suppression of plant defences will enable us to determine their role in shaping direct and indirect interactions in food webs and the extent to which these determine the coexistence and persistence of species.     

Rapid evolution of an adaptive cyanogenesis cline in introduced North American white clover (Trifolium repens L.)

White clover is polymorphic for cyanogenesis (HCN production after tissue damage), and this herbivore defence polymorphism has served as a classic model for studying adaptive variation. The cyanogenic phenotype requires two interacting biochemical components; the presence/absence of each component is controlled by a simple Mendelian gene (Ac/ac and Li/li). Climate-associated cyanogenesis clines occur in both native (Eurasian) and introduced populations worldwide, with cyanogenic plants predominating in warmer locations. Moreover, previous studies have suggested that epistatic selection may act within populations to maintain cyanogenic (AcLi) plants and acyanogenic plants that lack both components (acli plants) at the expense of plants possessing a single component (Acli and acLi plants). Here, we examine the roles of selection, gene flow and demography in the evolution of a latitudinal cyanogenesis cline in introduced North American populations. Using 1145 plants sampled across a 1650 km transect, we determine the distribution of cyanogenesis variation across the central United States and investigate whether clinal variation is adaptive or an artefact of population introduction history. We also test for the evidence of epistatic selection. We detect a clear latitudinal cline, with cyanogenesis frequencies increasing from 11% to 86% across the transect. Population structure analysis using nine microsatellite loci indicates that the cline is adaptive and not a by-product of demographic history. However, we find no evidence for epistatic selection within populations. Our results provide strong evidence for rapid adaptive evolution in these introduced populations, and they further suggest that the mechanisms maintaining adaptive variation may vary among populations of a species.     

Do Leaf Cutting Ants Cut Undetected? Testing the Effect of Ant-Induced Plant Defences on Foraging Decisions in Atta colombica

Leaf-cutting ants (LCAs) are polyphagous, yet highly selective herbivores. The factors that govern their selection of food plants, however, remain poorly understood. We hypothesized that the induction of anti-herbivore defences by attacked food plants, which are toxic to either ants or their mutualistic fungus, should significantly affect the ants' foraging behaviour. To test this "induced defence hypothesis," we used lima bean (Phaseolus lunatus), a plant that emits many volatile organic compounds (VOCs) upon herbivore attack with known anti-fungal or ant-repellent effects. Our results provide three important insights into the foraging ecology of LCAs. First, leaf-cutting by Atta ants can induce plant defences: Lima bean plants that were repeatedly exposed to foraging workers of Atta colombica over a period of three days emitted significantly more VOCs than undamaged control plants. Second, the level to which a plant has induced its anti-herbivore defences can affect the LCAs' foraging behaviour: In dual choice bioassays, foragers discriminated control plants from plants that have been damaged mechanically or by LCAs 24 h ago. In contrast, strong induction levels of plants after treatment with the plant hormone jasmonic acid or three days of LCA feeding strongly repelled LCA foragers relative to undamaged control plants. Third, the LCA-specific mode of damaging leaves allows them to remove larger quantities of leaf material before being recognized by the plant: While leaf loss of approximately 15% due to a chewing herbivore (coccinelid beetle) was sufficient to significantly increase VOC emission levels after 24 h, the removal of even 20% of a plant's leaf area within 20 min by LCAs did not affect its VOC emission rate after 24 h. Taken together, our results support the "induced defence hypothesis" and provide first empirical evidence that the foraging behaviour of LCAs is affected by the induction of plant defence responses.     

Plant-mediated effects in the Brassicaceae on the performance and behaviour of parasitoids

Direct and indirect plant defences are well studied, particularly in the Brassicaceae. Glucosinolates (GS) are secondary plant compounds characteristic in this plant family. They play an important role in defence against herbivores and pathogens. Insect herbivores that are specialists on brassicaceous plant species have evolved adaptations to excrete or detoxify GS. Other insect herbivores may even sequester GS and employ them as defence against their own antagonists, such as predators. Moreover, high levels of GS in the food plants of non-sequestering herbivores can negatively affect the growth and survival of their parasitoids. In addition to allelochemicals, plants produce volatile chemicals when damaged by herbivores. These herbivore induced plant volatiles (HIPV) have been demonstrated to play an important role in foraging behaviour of insect parasitoids. In addition, biosynthetic pathways involved in the production of HIPV are being unraveled using the model plant Arabidopsis thialiana. However, the majority of studies investigating the attractiveness of HIPV to parasitoids are based on experiments mainly using crop plant species in which defence traits may have changed through artificial selection. Field studies with both cultivated and wild crucifers, the latter in which defence traits are intact, are necessary to reveal the relative importance of direct and indirect plant defence strategies on parasitoid and plant fitness. Future research should also consider the potential conflict between direct and indirect plant defences when studying the evolution of plant defences against insect herbivory.     

Dual benefit from a belowground symbiosis: nitrogen fixing rhizobia promote growth and defense against a specialist herbivore in a cyanogenic plant

Legume-associated nitrogen-fixing bacteria play a key role for plant performance and productivity in natural and agricultural ecosystems. Although this plant-microbe mutualism has been known for decades, studies on effects of rhizobia colonisation on legume-herbivore interactions are scarce. We hypothesized that additional nitrogen provided by rhizobia may increase plant resistance by nitrogen-based defense mechanisms. We studied this below-aboveground interaction using a system consisting of lima bean (Phaseolus lunatus L.), rhizobia, and the Mexican bean beetle (Epilachna varivestis Muls.) as an insect herbivore. We showed that the rhizobial symbiosis not only promotes plant growth but also improves plant defense and resistance against herbivores. Results of our study lead to the suggestion that nitrogen provided by rhizobia is allocated to the production of nitrogen-containing cyanogenic defense compounds, and thereby crucially determines the outcome of plant-herbivore interactions. Our study supports the view that the fitness benefit of root symbioses includes defence mechanisms and thus extends beyond the promotion of plant growth. Since the associations between legumes and nitrogen-fixing rhizobia are ubiquitous in terrestrial ecosystems, improved knowledge on rhizobia-mediated effects on plant traits?Dand the resulting effects on higher trophic levels?Dis important for better understanding of the role of these microbes for ecosystem functioning.