The specificity of woody plant defense responses to different attacking herbivores is poorly known. We investigated the responses of black poplar ( Populus nigra ) to leaf feeding by three lepidopteran species ( Lymantria dispar, Laothoe populi and Amata mogadorensis ) and two leaf beetle species ( Phratora vulgatissima and Chrysomela populi ). Of the direct defenses monitored, increases in trypsin protease inhibitor activity and the salicinoid salicin were triggered by herbivore damage, but this was not herbivore-specific. Moreover, the majority of leaf salicinoid content was present constitutively and not induced by herbivory. On the other hand, volatile emission profiles did vary among herbivore species, especially between coleopterans and lepidopterans. Monoterpenes and sesquiterpenes were induced in damaged and adjacent undamaged leaves, while the emission of green leaf volatiles, aromatic and nitrogen-containing compounds (known to attract herbivore enemies) was restricted to damaged leaves. In conclusion, indirect defenses appear to show more specific responses to attacking herbivores than direct defenses in this woody plant. Electronic supplementary material The online version of this article (10.1007/s10886-019-01050-y) contains supplementary material, which is available to authorized users.

Background Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. Results We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. Conclusions We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.