unpredictable, ranging anywhere between 0 to .90% [23]. Promoting plant attractiveness to natural enemies is a novel alternative to traditional broad-spectrum pesticides, which indis- criminately kill predators and parasitoids and often lead to subsequent pest resurgence [24–27]. Deploying herbivore-induced plant volatiles (HIPVs) aboveground by controlled release dispensers has been shown to increase recruitment and retention of beneficial natural enemies to plants [27–29]. In an analogous belowground investigation, EPN infection of western corn root- worm (Diabrotica virgifera virgifera LeConte) larvae was increased by spiking soil surrounding maize roots with the HIPV, (E)-b- caryophyllene [11]. Herein, we investigated the mechanisms by which a novel HIPV affects naturally occurring EPN species and their consequence on belowground herbivores in two distinct agroecosystems. We have recently shown that a citrus root stock (Citrus paradisi Macf. 6 Poncirus trifoliata L. Raf.) releases HIPVs in response to larval feeding by the weevil, D. abbreviatus, and that these HIPVs attract EPN species in lab bioassays [7,8]. Here we characterize the specific HIPV attractant as 1,5-dimethylcyclodeca-1,5,7-triene (pregeijerene) and show its real-time release in response to herbivory. We also demonstrate that field application of this volatile increases mortality of belowground root weevils by attracting naturally occurring nematodes. We used recently developed qPCR primers and probes to detect and enumerate cryptic species of EPNs allowing for species-specific quantification of nematode response to attractants belowground. Given the broad effect of pregeijerene on EPN species, we also tested and demonstrated its efficacy in a non-citrus, temperate climate agroecosystem. The use of plant-produced signals, such as the damage-induced release of pregeijerene, along with conservation biological control strategies, could extend the usefulness of EPNs in crops damaged by belowground herbivores. Results Induction of Root Volatiles Volatiles were non-destructively sampled every three h from the root zone of citrus seedlings in glass chambers (Analytical Research Systems, Gainesville, FL) with sandy soil [8]. Gas chromatography-mass spectrometry (GC-MS) revealed 1,5-di- methylcyclodeca-1,5,7-triene (pregeijerene) as the dominating volatile, reaching a maximum release between nine and 12 h after initiation of larval feeding (Figure 1). There was an effect of treatment (F 1,4 = 26.4, P = 0.0005) and volatile release over time (F 3,2 = 2812, P = 0.0005). A stainless steel probe (Figure S3) was designed to collect volatiles in the field from the soil beds surrounding citrus trees in an unmanaged orchard. Here, GC-MS analyses again revealed pregeijerene in the root zone, as the most abundant volatile at one m away from the trunks of trees and still at detectable levels at 10 m from trees (Figure S4). Nematode Attraction in the Field using HIPVs We next conducted field tests to determine whether application of volatiles collected from infested roots would impact EPN- inflicted mortality of sentinel D. abbreviatus larvae. Commercially available EPN had been applied to the test orchard at numerous occasions; however, their persistence was not monitored. Cylin- drical mesh cages containing a single D. abbreviatus larva in autoclaved sandy soil [30] were treated with: (i) volatiles collected from weevil-infested roots or (ii) a blank solvent control (Figure S5). Larval mortality was 7466.9% in the presence of volatiles from infested roots, but only 4167.5% in the solvent alone treatment (N = 10, t18 = 2.75, P = 0.013). Subterranean Volatile Increases Biological Control Nematode Attraction in the Field using Isolated Pregeijerene A second experiment tested whether pregeijerene alone would increase mortality of larvae by attracting EPNs. For these experiments, a sufficient amount of pregeijerene was first extracted and purified from the roots of Common Rue (Ruta graveolens L.). The structure of extracted pregeijerene was confirmed by nuclear magnetic resonance (NMR) (Table S1, Table S2). To test for the attractiveness of pregeijerene, we first used serial dilutions of purified compound in dichloromethane in two-choice, sand-filled olfactometers [7,8,31]; 8 ng/ml (in 30 ml aliquots) was found to be the optimally attractive dosage to EPNs (S. riobrave and H. indica) (Figure 2). We used real-time qPCR to quantify the attraction of naturally occurring EPNs in the field and identified them to species. Our approach was to use species-specific primers and probes to identify EPN species known to either naturally occur in Florida: S. diaprepesi, H. indica, H. zealandica, and Steinernema sp. LWD1 (an undescribed species in the S. glaseri group); those which were applied to citrus orchards in the form of commercial biopesticides (S. riobrave); or those which might be introduced from natural long-distance spread from pastures and golf courses to manage mole crickets (S. scapterisci) [32,33]. Mortality of larvae buried with purified pregeijerene was . 3-fold higher than that of larvae buried with the solvent control (Figure 3A). The number of EPNs detected within (Figure 3B) and around (Figure 3C) cages containing purified pregeijerene was significantly higher than that from cages with the solvent control. Tukey HSD test indicated H. indica and H. zealandica were more abundant than Steinernema sp. LWD1 and S. diaprepesi (P , 0.0001 in all comparisons); however, there were no differences in the relative representation of species between treated and control samples. Neither S. riobrave, nor S. scapterisci were detected in any of the samples. Manipulation of Nematode Behavior with Pregeijerene in Blueberry Plantings Finally, we tested the generality of pregeijerene as an EPN attractant by deploying the compound in a geographically distant, non-citrus agricultural system: commercial highbush blueberry, Vaccinium corymbosum L., in Chatsworth, NJ. No pregeijerene was detected in volatiles collected from soil surrounding blueberry roots. Cages (described above) containing either a third-instar oriental beetle, Anomala orientalis Waterhouse, a scarab blueberry root pest or a late instar greater wax moth, Galleria mellonella L., larva (a widely used EPN sentinel), were deployed in blueberry fields. As described above, cages were treated with either blank solvent or pregeijerene. EPN-inflicted larval mortality (combined A. orientalis and G. mellonella) was nearly 2-fold greater in treatments with pregeijerene (55%) than those with solvent alone (30%) (P = 0.009). The increase was highly significant (from 40% to 80%) for G. mellonella (P = 0.003), but not statistically significant (from 20% to 30%) for A. orientalis (P = 0.552). Emerging EPNs were identified as S. glaseri with real- time PCR. On average, there were more S. glaseri nematodes surrounding the treatment (Mean6SE, 7.9662.91) than in the control (4.4362.56); however, this difference was not significant (N = 10, t18 = 0.909, P = 0.38). Discussion The obstacles of investigating belowground chemically mediat- ed interactions between plants and animals are being overcome gradually, opening opportunities for manipulating these interac- tions for enhanced biological control [34–36]. At least half of all plant biomass is attacked by underground herbivores and PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38146