Super-Q traps were replaced every three h for a 72-h period to track the time course of volatile release. The removed Super-Q traps were subsequently eluted with 150 ml of dichloromethane into individual 2.0 ml clear glass vials (Varian, Part Number 392611549, equipped with 500 ml glass inserts) [7]. The amount of pregeijerene detected over time for all treatments was subjected to multiple analysis of variance (MANOVA) to determine differences between, as well as, within treatments. It was a challenge to remove sufficient pregeijerene from infested roots for bioassays and field testing. However, it was previously established [56] that a hydrodistillate of Common Rue (Ruta graveolens) roots contained the related terpene, geijerene, as a major constituent (67% of the total volatile compounds). Pregeijerene easily converts to geijerene at temperatures exceeding 120uC [57] (Figure S1). On-column GC-MS analyses confirmed pregeijerene as the main naturally occurring terpene in roots of Common Rue that could be easily extracted and purified from crushed roots using a series of solid phase extractions (Figure S2). In situ Volatile Collection from Infested Roots in the Field Volatiles were collected from the soil beds surrounding citrus trees in the field. A soil probe (Figure S3) was used to sample soil volatiles at a depth of 20 cm and at distances of one and 10 m from the trunks of citrus trees. A vacuum pump was used to pull air at a rate of 200 ml/min for a total of 30 min. Compounds were collected on adsorbent traps filled with 50 mg of Super-Q attached to the top of the soil probe (Figure S3). The Super-Q traps were subsequently eluted as described in the previous section. Identification of Pregeijerene Pregeijerene isolated from Common Rue and that from citrus roots after herbivore feeding was identified by electron impact (EI) and chemical ionization (CI) GC-MS analyses on DB1, DB5, and DB35 GC columns. Although the EI mass spectra matched pregeijerene in the Adams 2 library, the lack of a standard made it necessary to confirm the structure by nuclear magnetic resonance (NMR) (described in Supporting Information Materials and Methods S1). Two-Choice Bioassay to Determine Optimal Dosage to Attract EPNs The behavioral responses of EPNs to collected pregeijerene were quantified in a two-choice, sand-filled olfactometer [7,31]. Briefly, the olfactometer consists of three detachable sections: two opposing 16-ml glass jars which contained treatments and a central connecting tube three cm in length with an apical hole into which EPN were applied. Dilutions from the purified R. graveolens root extract were placed on filter paper, which was allowed to dry for 30 s to allow solvent evaporation. Thereafter, filter papers were placed on the bottoms of each glass jar, which were then filled with moist (10% w/v) sterilized sand. The central chamber connecting the two arms of the olfactometer was also filled with sterilized and moistened sand. EPNs (ca. 200 IJs) were applied into the central orifice of the connecting tube and given eight h to respond. Following the incubation period, the column was disassembled and the IJs from the two collection jars were extracted using Baermann funnels. The experiment was replicated five times for each dilution and separately tested with two EPN species: S. riobrave Btw1 and H. indica Ker1. A student’s t-test was used to compare nematode response in the two-choice olfactometer. Since responses of both species to pregeijerene versus the solvent controls were identical, data for both species were combined prior to analysis (df = 18). The dosage Subterranean Volatile Increases Biological Control at which a significant proportion of EPNs were attracted to the treatment arm was selected for our field trial. Application of HIPVs in the Field An experiment was conducted in a sandy soil (97:2:1, sand:silt:clay; pH 7.1; 0.1% OM) citrus orchard at the CREC (28 07 26.84 N, 81 42 55.31 W). The experiment was placed within a section of mature orange trees spaced (without beds) 4.5 m within and 8.1 m between rows that was irrigated with microsprinklers. A randomized design was used to place treatments between trees in eight adjacent rows. Cylindrical wire-mesh cages containing autoclaved sandy soil (10% moisture) and a single D. abbreviatus larva (reared on artificial diet for 3 to 5 weeks) were buried 20 cm deep in the soil beneath the tree canopies. Cages were made of 225-mesh stainless-steel cylinders (7 length 6 3 cm diam) secured at each end with polypropylene snap-on caps. A replicate consisted of six cages placed equidis- tantly from one another in a circle pattern (48 cm diam) for each treatment. All cages contained a single D. abbreviatus larva and were baited with one of two treatments per replicate: (i) volatiles from roots fed upon by a D. abbreviatus larva or (ii) blank solvent control. There were 10 replicates of six cages per treatment. Treatments were applied as 30 ml aliquots to three cm diameter filter paper discs (Whatman). Solvent was allowed to evaporate for 30 s prior to insertion of filter papers at the base of each cage. The cages were left buried for 72 h. Eight soil core samples (2.5 cm diam 6 30 cm deep) were taken from soil surrounding the treatment arena before the cages were removed to measure the number of EPNs attracted to the surrounding treatment arena. Recovered larvae were rinsed and placed on moistened filter paper within individual Petri dishes to confirm EPN infection by subsequent infective juvenile emergence from cadavers. Mortality of the larvae caused by EPNs was recorded from 0 to 72 h after removal from soil. The effect of isolated pregeijerene on larval mortality was investigated in two additional experiments (one of which was conducted in a blueberry planting in Chatsworth, NJ, using A. oientalis and G. mellonella). The methods for these experiments were similar to those described above, except that the soil remaining within the six cages from each replication was placed in a container and homogenized for later nematode DNA extraction (n = 10). Soil cores taken from the surrounding treatment arenas were also combined and stored for nematode DNA extraction (n = 10). Fisher’s exact test was used to compare larval mortality between the treatment and control. Only soil samples from the citrus experiment were analyzed for DNA quantification. Detection, Identification, and Quantification of Entomopathogenic Nematodes Using Real-Time qPCR Real-time qPCR was used to quantify attraction of naturally occurring EPN species to volatiles applied in the field and to identify nematodes to species. This technique targeted 11 EPN species [32,33,44]. In the citrus experiment, we surveyed the natural occurrence of six species (S. diaprepesi, Steinernema sp. glaseri group, S. riobrave, S. scapterisci, H. indica, and H. zealandica); in the blueberry experiment, nine species were surveyed (S. carpocapsae, S. feltiae, S. glaseri, S. kraussei, S. scapterisci, Steinernema sp. glaseri group, H. indica, H. zealandica, and H. bacteriophora). Briefly, species-specific primers and TaqManH probes were designed from the ITS rDNA region using sequences of the target species as well as closely related species recovered from the NCBI database or generated by the authors in that study. Multiple alignments of the correspond- ing sequences were performed [58] to select areas of variability in the ITS region. The designed primers and probes provided no non-specific amplification when they were tested using other EPN PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38146