Plant metabolism and defence strategies in the flowering stage: Time-dependent responses of leaves and flowers under attack.

Plants developing into the flowering stage undergo major physiological changes. Because flowers are reproductive tissues and resource sinks, strategies to defend them may differ from those for leaves. Thus, this study investigates the defences of flowering plants by assessing processes that sustain resistance (constitutive and induced) and tolerance to attack. We exposed the annual plant Brassica nigra to three distinct floral attackers (caterpillar, aphid and bacterial pathogen) and measured whole-plant responses at 4, 8 and 12 days after the attack. We simultaneously analysed profiles of primary and secondary metabolites in leaves and inflorescences and measured dry biomass of roots, leaves and inflorescences as proxies of resource allocation and regrowth. Regardless of treatments, inflorescences contained 1.2 to 4 times higher concentrations of primary metabolites than leaves, and up to 7 times higher concentrations of glucosinolates, which highlights the plant's high investment of resources into inflorescences. No induction of glucosinolates was detected in inflorescences, but the attack transiently affected the total concentration of soluble sugars in both leaves and inflorescences. We conclude that B. nigra evolved high constitutive rather than inducible resistance to protect their flowers; plants additionally compensated for damage by attackers via the regrowth of reproductive parts. This strategy may be typical of annual plants.

Negative Effects of Rhizobacteria Association on Plant Recruitment of Generalist Predators.

Plant-associated microbes can influence above- and belowground interactions between plants and other organisms and thus have significant potential for use in the management of agricultural ecosystems. However, fully realizing this potential will require improved understanding of the specific ways in which microbes influence plant ecology, which are both more complex and less well studied than the direct effects of microbes on host-plant physiology. Microbial effects on mutualistic and antagonistic interactions between plants and insects are of particular interest in this regard. This study examines the effects of two strains of Pseudomonas rhizobacteria on the direct and indirect (predator-mediated) resistance of tomato plants to a generalist herbivore (Spodoptera littoralis) and associated changes in levels of defense compounds. We observed no significant effects of rhizobacteria inoculation on caterpillar weight, suggesting that rhizobacteria did not influence direct resistance. However, the generalist predator Podisus maculiventris avoided plants inoculated with one of our rhizobacteria strains, Pseudomonas simiae. Consistent with these results, we found that inoculation with P. simiae influenced plant volatile emissions, but not levels of defense-related compounds. These findings show that rhizobacteria can negatively affect the attraction of generalist predators, while highlighting the complexity and context dependence of microbial effects on plant-insect interactions.

Skin bacterial volatiles: propelling the future of vector control.

The skin microbiota plays an essential role in the protection against pathogens. It is our skin microbiota that makes us smell different from each other, rendering us more or less attractive to mosquitoes. Mosquitoes exploit skin bacterial odours to locate their hosts and are vectors of pathogens that can cause severe diseases such as malaria and dengue fever. A novel solution for long-lasting protection against insect vectors of disease could be attained by manipulating the bacterial commensals on human skin. The current options for protection against biting insects usually require topical application of repellents that evaporate within hours. We discuss possible routes for the use of commensal bacteria to create a microbial-based repellent.

Multiple Attack to Inflorescences of an Annual Plant Does Not Interfere with the Attraction of Parasitoids and Pollinators.

Plants in the flowering stage need to ensure reproduction by protecting themselves from attack and by preserving interactions with mutualist pollinators. When different plant mutualists are using the same type of cues, such as volatile compounds, attraction of parasitoids and pollinators may trade off. To explore this, we compared volatile emission of Brassica nigra plants in response to single or dual attack on their inflorescences. Additionally, we recorded flower visitation by pollinators and the attraction of parasitoids in the greenhouse and/or field. Brassica nigra were exposed in the flowering stage to one or two of the following three attackers: Brevicoryne brassicae aphids, Pieris brassicae caterpillars, and Xanthomonas campestris pv. raphani bacteria. We found that single attack by caterpillars, and dual attack by caterpillars plus aphids, induced the strongest changes in plant volatile emission. The caterpillars' parasitoid C. glomerata did not exhibit preference for plants exposed to caterpillars only vs. plants exposed to caterpillars plus aphids or plus bacteria. However, the composition of the pollinator community associated with flowers of B. nigra was affected by plant exposure to the attackers, but the total number of pollinators visiting the plants did not change upon attack. We conclude that, when B. nigra were exposed to single or dual attack on their inflorescences, the plants maintained interactions with natural enemies of the insect attackers and with pollinators. We discuss how chemical diversity may contribute to plant resilience upon attack.

Volatiles from soil-borne fungi affect directional growth of roots.

Volatiles play major roles in mediating ecological interactions between soil (micro)organisms and plants. It is well-established that microbial volatiles can increase root biomass and lateral root formation. To date, however, it is unknown whether microbial volatiles can affect directional root growth. Here, we present a novel method to study belowground volatile-mediated interactions. As proof-of-concept, we designed a root Y-tube olfactometer, and tested the effects of volatiles from four different soil-borne fungi on directional growth of Brassica rapa roots in soil. Subsequently, we compared the fungal volatile organic compounds (VOCs) previously profiled with Gas Chromatography-Mass Spectrometry (GC-MS). Using our newly designed setup, we show that directional root growth in soil is differentially affected by fungal volatiles. Roots grew more frequently toward volatiles from the root pathogen Rhizoctonia solani, whereas volatiles from the other three saprophytic fungi did not impact directional root growth. GC-MS profiling showed that six VOCs were exclusively emitted by R. solani. These findings verify that this novel method is suitable to unravel the intriguing chemical cross-talk between roots and soil-borne fungi and its impact on root growth.

Plant ontogeny determines strength and associated plant fitness consequences of plant-mediated interactions between herbivores and flower visitors.

Plants show ontogenetic variation in growth-defence strategies to maximize reproductive output within a community context. Most work on plant ontogenetic variation in growth-defence trade-offs has focussed on interactions with antagonistic insect herbivores. Plants respond to herbivore attack with phenotypic changes. Despite the knowledge that plant responses to herbivory affect plant mutualistic interactions with pollinators required for reproduction, indirect interactions between herbivores and pollinators have not been included in the evaluation of how ontogenetic growth-defence trajectories affect plant fitness.In a common garden experiment with the annual Brassica nigra, we investigated whether exposure to various herbivore species on different plant ontogenetic stages (vegetative, bud or flowering stage) affects plant flowering traits, interactions with flower visitors and results in fitness consequences for the plant.Effects of herbivory on flowering plant traits and interactions with flower visitors depended on plant ontogeny. Plant exposure in the vegetative stage to the caterpillar Pieris brassicae and aphid Brevicoryne brassicae led to reduced flowering time and flower production, and resulted in reduced pollinator attraction, pollen beetle colonization, total seed production and seed weight. When plants had buds, infestation by most herbivore species tested reduced flower production and pollen beetle colonization. Pollinator attraction was either increased or reduced. Plants infested in the flowering stage with P. brassicae or Lipaphis erysimi flowered longer, while infestation by any of the herbivore species tested increased the number of flower visits by pollinators.Our results show that the outcome of herbivore-flower visitor interactions in B. nigra is specific for the combination of herbivore species and plant ontogenetic stage. Consequences of herbivory for flowering traits and reproductive output were strongest when plants were attacked early in life. Such differences in selection pressures imposed by herbivores to specific plant ontogenetic stages may drive the evolution of distinct ontogenetic trajectories in growth-defence-reproduction strategies and include indirect interactions between herbivores and flower visitors. Synthesis. Plant ontogeny can define the direct and indirect consequences of herbivory. Our study shows that the ontogenetic stage of plant individuals determined the effects of herbivory on plant flowering traits, interactions with flower visitors and plant fitness.

Settling on leaves or flowers: herbivore feeding site determines the outcome of indirect interactions between herbivores and pollinators.

Herbivore attack can alter plant interactions with pollinators, ranging from reduced to enhanced pollinator visitation. The direction and strength of effects of herbivory on pollinator visitation could be contingent on the type of plant tissue or organ attacked by herbivores, but this has seldom been tested experimentally. We investigated the effect of variation in feeding site of herbivorous insects on the visitation by insect pollinators on flowering Brassica nigra plants. We placed herbivores on either leaves or flowers, and recorded the responses of two pollinator species when visiting flowers. Our results show that variation in herbivore feeding site has profound impact on the outcome of herbivore-pollinator interactions. Herbivores feeding on flowers had consistent positive effects on pollinator visitation, whereas herbivores feeding on leaves did not. Herbivores themselves preferred to feed on flowers, and mostly performed best on flowers. We conclude that herbivore feeding site choice can profoundly affect herbivore-pollinator interactions and feeding site thereby makes for an important herbivore trait that can determine the linkage between antagonistic and mutualistic networks.

No evidence of flowering synchronization upon floral volatiles for a short lived annual plant species: revisiting an appealing hypothesis.

BACKGROUND: Self-incompatible plants require simultaneous flowering mates for crosspollination and reproduction. Though the presence of flowering conspecifics and pollination agents are important for reproductive success, so far no cues that signal the flowering state of potential mates have been identified. Here, we empirically tested the hypothesis that plant floral volatiles induce flowering synchrony among self-incompatible conspecifics by acceleration of flowering and flower opening rate of non-flowering conspecifics. We exposed Brassica rapa Maarssen, a self-incompatible, in rather dense patches growing annual, to (1) flowering or non-flowering conspecifics or to (2) floral volatiles of conspecifics by isolating plants in separate containers with a directional airflow. In the latter, odors emitted by non-flowering conspecifics were used as control. RESULTS: Date of first bud, duration of first flower bud, date of first flower, maximum number of open flowers and flower opening rate were not affected by the presence of conspecific flowering neighbors nor by floral volatiles directly. CONCLUSIONS: This study presents a compelling approach to empirically test the role of flower synchronization by floral volatiles and challenges the premises that are underlying this hypothesis. We argue that the life history of the plant as well as its interaction with pollinators and insect herbivores, as well as the distance over which volatiles may serve as synchronization cue, set constraints on the fitness benefits of synchronized flowering which needs to be taken into account when testing the role of floral volatiles in synchronized flowering.

Ecology of Plastic Flowers.

Plant phenotypic plasticity in response to herbivore attack includes changes in flower traits. Such herbivore-induced changes in flower traits have consequences for interactions with flower visitors. We synthesize here current knowledge on the specificity of herbivore-induced changes in flower traits, the underlying molecular mechanisms, and the ecological consequences for flower-associated communities. Herbivore-induced changes in flower traits seem to be largely herbivore species-specific. The extensive plasticity observed in flowers influences a highly connected web of interactions within the flower-associated community. We argue that the adaptive value of herbivore-induced plant responses and flower plasticity can be fully understood only from a community perspective rather than from pairwise interactions.

Volatiles of pathogenic and non-pathogenic soil-borne fungi affect plant development and resistance to insects.

Plants are ubiquitously exposed to a wide diversity of (micro)organisms, including mutualists and antagonists. Prior to direct contact, plants can perceive microbial organic and inorganic volatile compounds (hereafter: volatiles) from a distance that, in turn, may affect plant development and resistance. To date, however, the specificity of plant responses to volatiles emitted by pathogenic and non-pathogenic fungi and the ecological consequences of such responses remain largely elusive. We investigated whether Arabidopsis thaliana plants can differentiate between volatiles of pathogenic and non-pathogenic soil-borne fungi. We profiled volatile organic compounds (VOCs) and measured CO2 emission of 11 fungi. We assessed the main effects of fungal volatiles on plant development and insect resistance. Despite distinct differences in VOC profiles between the pathogenic and non-pathogenic fungi, plants did not discriminate, based on plant phenotypic responses, between pathogenic and non-pathogenic fungi. Overall, plant growth was promoted and flowering was accelerated upon exposure to fungal volatiles, irrespectively of fungal CO2 emission levels. In addition, plants became significantly more susceptible to a generalist insect leaf-chewing herbivore upon exposure to the volatiles of some of the fungi, demonstrating that a prior fungal volatile exposure can negatively affect plant resistance. These data indicate that plant development and resistance can be modulated in response to exposure to fungal volatiles.

Floral plasticity: Herbivore-species-specific-induced changes in flower traits with contrasting effects on pollinator visitation.

Plant phenotypic plasticity in response to antagonists can affect other community members such as mutualists, conferring potential ecological costs associated with inducible plant defence. For flowering plants, induction of defences to deal with herbivores can lead to disruption of plant-pollinator interactions. Current knowledge on the full extent of herbivore-induced changes in flower traits is limited, and we know little about specificity of induction of flower traits and specificity of effect on flower visitors. We exposed flowering Brassica nigra plants to six insect herbivore species and recorded changes in flower traits (flower abundance, morphology, colour, volatile emission, nectar quantity, and pollen quantity and size) and the behaviour of two pollinating insects. Our results show that herbivory can affect multiple flower traits and pollinator behaviour. Most plastic floral traits were flower morphology, colour, the composition of the volatile blend, and nectar production. Herbivore-induced changes in flower traits resulted in positive, negative, or neutral effects on pollinator behaviour. Effects on flower traits and pollinator behaviour were herbivore species-specific. Flowers show extensive plasticity in response to antagonist herbivores, with contrasting effects on mutualist pollinators. Antagonists can potentially act as agents of selection on flower traits and plant reproduction via plant-mediated interactions with mutualists.

Caterpillars induce jasmonates in flowers and alter plant responses to a second attacker.

In nature, herbivorous insects and plant pathogens are generally abundant when plants are flowering. Thus, plants face a diversity of attackers during their reproductive phase. Plant responses to one attacker can interfere with responses to a second attacker, and phytohormones that orchestrate plant reproduction are also involved in resistance to insect and pathogen attack. We quantified phytohormonal responses of flowering plants exposed to single or dual attack and studied resistance mechanisms of plants in the flowering stage. Flowering Brassica nigra were exposed to either a chewing caterpillar, a phloem-feeding aphid or a bacterial pathogen, and plant hormonal responses were compared with dual attack situations. We quantified phytohormones in inflorescences and leaves, and determined the consequences of hormonal changes for components of direct and indirect plant resistance. Caterpillars were the main inducers of jasmonates in inflorescences, and the phytohormonal profile of leaves was not affected by either insect or pathogen attack. Dual attack increased plant resistance to caterpillars, but compromised resistance to aphids. Parasitoid performance was negatively correlated with the performance of their hosts. We conclude that plants prioritize resistance of reproductive tissues over vegetative tissues, and that a chewing herbivore species is the main driver of responses in flowering B. nigra.

Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications.

Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta-analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals.

Plant Phenotypic and Transcriptional Changes Induced by Volatiles from the Fungal Root Pathogen Rhizoctonia solani.

Beneficial soil microorganisms can affect plant growth and resistance by the production of volatile organic compounds (VOCs). Yet, little is known on how VOCs from soil-borne plant pathogens affect plant growth and resistance. Here we show that VOCs released from mycelium and sclerotia of the fungal root pathogen Rhizoctonia solani enhance growth and accelerate development of Arabidopsis thaliana. Seedlings briefly exposed to the fungal VOCs showed similar phenotypes, suggesting that enhanced biomass and accelerated development are primed already at early developmental stages. Fungal VOCs did not affect plant resistance to infection by the VOC-producing pathogen itself but reduced aboveground resistance to the herbivore Mamestra brassicae. Transcriptomics of A. thaliana revealed that genes involved in auxin signaling were up-regulated, whereas ethylene and jasmonic acid signaling pathways were down-regulated by fungal VOCs. Mutants disrupted in these pathways showed similar VOC-mediated growth responses as the wild-type A. thaliana, suggesting that other yet unknown pathways play a more prominent role. We postulate that R. solani uses VOCs to predispose plants for infection from a distance by altering root architecture and enhancing root biomass. Alternatively, plants may use enhanced root growth upon fungal VOC perception to sacrifice part of the root biomass and accelerate development and reproduction to survive infection.

Integrating Studies on Plant-Pollinator and Plant-Herbivore Interactions.

Research on herbivore-induced plant defence and research on pollination ecology have had a long history of separation. Plant reproduction of most angiosperm species is mediated by pollinators, and the effects of herbivore-induced plant defences on pollinator behaviour have been largely neglected. Moreover, there is expected to be a trade-off between plant reproductive strategies and defence mechanisms. To investigate this trade-off, it is essential to study herbivore-induced plant resistance and allocation of resources by plants, within the same system, and to test if indirect plant resistance can conflict with pollinator attraction. Here, I review the key literature highlighting connection between plant defence and reproduction, and propose to exploit natural variation among plant species to assess the ecological costs of plant responses to herbivores and pollinators.

Caught between parasitoids and predators - survival of a specialist herbivore on leaves and flowers of mustard plants.

The survival of insect herbivores typically is constrained by food choice and predation risk. Here, we explored whether movement from leaves to flowers increases survival of herbivores that prefer to feed on floral tissues. Combining field and greenhouse experiments, we investigated whether flowering influences the behavior of Pieris brassicae butterflies and caterpillars and, consequently, herbivore survival in the field. In this context, we investigated also if flowers of Brassica nigra can provide caterpillars refuge from the specialist parasitoid Cotesia glomerata and from predatory social wasps. By moving to flowers, caterpillars escaped from the parasitoid. Flowers are nutritionally superior when compared with leaves, and caterpillars develop faster when feeding on flowers. However, late-stage caterpillars can be preyed upon intensively by social wasps, irrespective of whether they feed on leaves or flowers. We conclude that flower preference by P. brassicae is more likely driven by nutritional advantages and reduced parasitism on flowers, than by risks of being killed by generalist predators.

Folivory affects composition of nectar, floral odor and modifies pollinator behavior.

Herbivory induces changes in plants that influence the associated insect community. The present study addresses the potential trade-off between plant phytochemical responses to insect herbivory and interactions with pollinators. We used a multidisciplinary approach and have combined field and greenhouse experiments to investigate effects of herbivory in plant volatile emission, nectar production, and pollinator behavior, when Pieris brassicae caterpillars were allowed to feed only on the leaves of Brassica nigra plants. Interestingly, volatile emission by flowers changed upon feeding by herbivores on the leaves, whereas, remarkably, volatile emission by leaves did not significantly differ between infested and non-infested flowering plants. The frequency of flower visits by pollinators was generally not influenced by herbivory, but the duration of visits by honeybees and butterflies was negatively affected by herbivore damage to leaves. Shorter duration of pollinator visits could be beneficial for a plant, because it sustains pollen transfer between flowers while reducing nectar consumption per visit. Thus, no trade-off between herbivore-induced plant responses and pollination was evident. The effects of herbivore-induced plant responses on pollinator behavior underpin the importance of including ecological factors, such as herbivore infestation, in studies of the ecology of plant pollination.

Phenotypic plasticity of plant response to herbivore eggs: effects on resistance to caterpillars and plant development.

Herbivory induces direct resistance responses in plants that negatively affect subsequently colonizing herbivores. Moreover, eggs of herbivorous insects can also activate plant resistance, which in some cases prevents hatching larvae from feeding. Until now, plant-mediated effects of eggs on subsequent herbivory, and the specificity of such responses, have remained poorly understood. We studied the specificity and effects of plant resistance induced by herbivore egg deposition against lepidopteran larvae of species with different dietary breadths, feeding on a wild annual plant, the crucifer Brassica nigra. We examined whether this plant-mediated response affects the growth of caterpillars of a specialist (Pieris brassicae) that feeds on B. nigra leaves and flowers, and a generalist (Mamestra brassicae) that rarely attacks this wild crucifer. We measured growth rates of neonate larvae to the end of their second instar after the larvae had hatched on plants exposed to eggs vs. plants without eggs, under laboratory and semi-field conditions. Moreover, we studied the effects of egg deposition by the two herbivore species on plant height and flowering rate before and after larval hatching. Larvae of both herbivore species that developed on plants previously infested with eggs of the specialist butterfly P. brassicae gained less mass compared with larvae that developed on egg-free plants. Plants exposed to butterfly eggs showed accelerated plant growth and flowering compared to egg-free plants. Egg deposition by the generalist moth M. brassicae, in contrast, had no effect on subsequent performance by either herbivore species, or on plant development. Our results demonstrate that B. nigra plants respond differently to eggs of two herbivore species in terms of plant development and induced resistance to caterpillar attack. For this annual crucifer, the retardation of caterpillar growth in response to deposition of eggs by P. brassicae in combination with enhanced growth and flowering likely result in reproductive assurance, after being exposed to eggs from an herbivore whose larvae rapidly reduce the plant's reproductive potential through florivory.