Plants interact with a multitude of microorganisms and insects, both belowand above ground, which might influence plant metabolism. Despite this, we lack knowledge of the impact of natural soil communities and multiple aboveground attackers on the metabolic responses of plants, and whether plant metabolic responses to single attack can predict responses to dual attack. We used untargeted metabolic fingerprinting (gas chromatographymass spectrometry, GC-MS) on leaves of the pedunculate oak, Quercus robur, to assess the metabolic response to different soil microbiomes and aboveground single and dual attack by oak powdery mildew (Erysiphe alphitoides) and the common oak aphid (Tuberculatus annulatus). Distinct soil microbiomes were not associated with differences in the metabolic profile of oak seedling leaves. Single attacks by aphids or mildew had pronounced but different effects on the oak leaf metabolome, but we detected no difference between the metabolomes of healthy seedlings and seedlings attacked by both aphids and powdery mildew. Our findings show that aboveground attackers can have species-specific and non-additive effects on the leaf metabolome of oak. The lack of a metabolic signature detected by GC-MS upon dual attack might suggest the existence of a potential negative feedback, and highlights the importance of considering the impacts of multiple attackers to gain mechanistic insights into the ecology and evolution of species interactions and the structure of plant-associated communities, as well as for the development of sustainable strategies to control agricultural pests and diseases and plant breeding.

This dataset contains the metabolite annotation of Quercus robur leaves under single or dual attack (by aphids and powdery mildew), obtained by GCMS.

At SMC, sample preparation for GC-MS was performed according to Gullberg et al. (2004). More specifically, 750 µL of extraction buffer (20/20/60 v/v/v chloroform:water:methanol) including internal standards were added to each sample. The sample was shaken with a tungsten bead in a mixer mill at 30 Hz for 3 min, after which the bead was removed and the sample was centrifuged at 4◦C and 14,000 rpm, for 10 min. 200 µL of supernatant were transferred to a microvial and solvents were evaporated. Derivatization was performed according to Gullberg et al. (2004). More specifically, 30 µL of methoxyamine (15 µg/µL in pyridine) were added to the dry sample and the sample was shaken vigorously for 10 min before being left to react in room temperature. After 16 h, 30 µL of MSTFA and 30 µL of heptane were added and the sample was shaken and left to react for 1 h in room temperature. 30 µL of methyl stearate (15 ng/µL in heptane) were added before analysis.