Subterranean chemical ecology of tritrophic interactions: Citrus roots, roots weevils and entomopathogenic nematodes

In response to herbivore feeding, plants release odors that benefit them by attracting natural enemies of herbivorous insects. Such interactions have been thoroughly examined aboveground. It has become increasingly evident that similar interactions occur belowground. The root-weevil (Diaprepes abbreviatus) is a serious citrus pest. Entomopathogenic nematodes (EPNs) have varying, and unpredictable, efficacy in controlling the weevil. Interactions between the plant, insect and nematode are poorly understood. In root-zone bioassays, root-weevil infested rootstock (Swingle) recruited significantly more EPNs than non-infested or mechanically damaged roots, or larvae alone. GC-MS analysis detected unique volatiles released from roots in response to weevil feeding. We compared attraction to volatiles of infested and non-infested roots from the hybrid, Swingle rootstock, and a parent line of the hybrid, P. trifoliata (Pt). Volatiles from Swingle infested by weevils were more attractive to both EPNs and plant parasitic nematodes than non-infested roots irrespective of foraging strategy. Pt, attracted EPN species irrespective of insect herbivory. Analysis of root volatiles revealed that Pt released the attractive cue constitutively, regardless of weevil feeding. A different non-hybrid species (C. aurantium) released the attractive cue only in response to larval feeding. Pregeijerene (1, 5-dimethylcyclodeca-1, 5, 7-triene) was identified as the major constituent of EPN attraction released from weevil-damaged roots. The release of pregeijerene by citrus roots peaked 9-12 hr after initiation of larval root feeding. Volatile collections from above/belowground portions of citrus plants revealed that aboveground adult feeding does not induce production of pregeijerene analogous to that induced by root damage nor does damage by larvae belowground induce a similar cue aboveground. Through the development of novel in-situ volatile sampling methods, pregeijerene release was detected from roots of mature trees in the field. In field experiments, lab-collected citrus volatiles from infested roots and isolated pregeijerene increased mortality of beetle larvae compared with controls. Using species-specific probes designed to identify EPN species, we determined by quantitative real-time PCR that field application of pregeijerene increased pest mortality by attracting four species of EPNs native to Florida. This and similar chemicals may have broad application for controlling agriculturally significant root pests.