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.

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.

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.

Terpenoid biosynthesis in Arabidopsis attacked by caterpillars and aphids: effects of aphid density on the attraction of a caterpillar parasitoid.

One of the responses of plants to insect attack is the production of volatile organic compounds that mediate indirect defence of plants by attracting natural enemies of the attacking herbivores. Herbivore-induced plant volatiles (HIPVs) include terpenoids that play key roles in the attraction of natural enemies. Crosstalk between phytohormonal signalling pathways is well known to affect the regulation of plant defences, including the emission of HIPVs. Thus, simultaneous feeding on the same plant by caterpillars and aphids, can affect the attraction of parasitoids by the plant compared to single insect attack. The role of aphid density in the regulation of HIPV emission by plants under dual attack has not been studied previously. Here, we investigated the attraction of Diadegma semiclausum, a parasitoid of the Diamondback moth Plutella xylostella, to volatiles emitted by Arabidopsis thaliana plants, simultaneously attacked by host caterpillars, and by the non-host aphid Brevicoryne brassicae. Our study shows that the effect of aphid infestation on parasitoid attraction is influenced by the density of the aphids. Biosynthesis and emission of (E,E)-α-farnesene could be linked to the observed preference of D. semiclausum parasitoids for the HIPV blend emitted by plants dually infested by caterpillars and aphids at a high density compared to dually infested plants with a low aphid density. Parasitoids such as D. semiclausum are important enemies of herbivorous insects and a better understanding of how plants express indirect defence mechanisms in response to multiple insect attack will provide important knowledge on plant-herbivore-parasitoid interactions under multiple stress conditions.

An invasive insect herbivore disrupts plant volatile-mediated tritrophic signalling.

Plants respond to insect attack by emission of volatile organic compounds, which recruit natural enemies of the attacking herbivore, constituting an indirect plant defence strategy. In this context, the egg parasitoid Trissolcus basalis is attracted by oviposition-induced plant volatiles emitted by Vicia faba plants as a consequence of feeding and oviposition by the pentatomid host Nezara viridula. However, this local tritrophic web could be affected by the recent invasion by the alien pentatomid bug Halyomorpha halys, an herbivore that shares the same environments as native pentatomid pests. Therefore, we investigated in laboratory conditions the possible impact of H. halys on the plant volatile-mediated signalling in the local tritrophic web V. faba-N. viridula-T. basalis. We found that T. basalis wasps were not attracted by volatiles induced in the plants by feeding and oviposition activities of H. halys, indicating specificity in the wasps' response. However, the parasitoid attraction towards plant volatiles emitted as a consequence of feeding and oviposition by the associated host was disrupted when host, N. viridula, and non-associated host, H. halys, were concurrently present on the same plant, indicating that invasion by the alien herbivore interferes with established semiochemical webs. These outcomes are discussed in a context of multiple herbivory by evaluating the possible influences of alien insects on local parasitoid foraging behaviour.