Removing soil confounds: a double–cup hydroponic system for in situ study of belowground plant–herbivore interactions

Studying belowground plant-herbivore interactions presents significant methodological challenges, as soil organic matter can serve as an alternative food source for root-feeding herbivores, confounding the assessment of treatment effects. To address these limitations, we developed and validated a novel double-cup hydroponic air-chamber system that enables in situ evaluation of root herbivory without disrupting plant nutrition or introducing soil-derived confounds. Using this platform, we implemented a fully factorial design including plants with and without the endophyte Epichloë pampeana, with and without silicon (Si) supplementation, and with and without root herbivory by white grubs, using the cool-season grass Bromus auleticus. The system proved highly effective, maintaining larval viability and active root-feeding throughout the assay without requiring root excision. Herbivory significantly reduced the root-to-shoot (R:S) ratio, indicating severe structural damage. Although neither Si supplementation nor endophytic status significantly affected insect performance or plant tolerance under these conditions, the system represents a robust experimental platform. By ensuring physiological stability and precise nutritional control, it provides new opportunities to investigate belowground ecology and plant-herbivore signaling under strictly controlled conditions.