Subterranean, Herbivore-Induced Plant Volatile Increases Biological Control Activity of Multiple Beneficial Nematode Species in Distinct Habitats Jared G. Ali1¤, Hans T. Alborn2, Raquel Campos-Herrera1,3, Fatma Kaplan2, Larry W. Duncan1, Cesar Rodriguez-Saona4, Albrecht M. Koppenho¨ fer4, Lukasz L. Stelinski1* 1 Entomology and Nematology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, Florida, United States of America, 2 Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, Florida, United States of America, 3 Departamento de Contaminaci o´n Ambiental, Instituto de Ciencias Agrarias, Centro de Ciencias Medioambientales, Madrid, Spain, 4 Department of Entomology, Rutgers University, New Brunswick, New Jersey, United States of America Abstract While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well documented aboveground, new evidence suggests that belowground volatile emissions can protect plants by attracting entomopathogenic nematodes (EPNs). However, due to methodological limitations, no study has previously detected belowground herbivore-induced volatiles in the field or quantified their impact on attraction of diverse EPN species. Here we show how a belowground herbivore-induced volatile can enhance mortality of agriculturally significant root pests. First, in real time, we identified pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene) from citrus roots 9–12 hours after initiation of larval Diaprepes abbreviatus feeding. This compound was also detected in the root zone of mature citrus trees in the field. Application of collected volatiles from weevil-damaged citrus roots attracted native EPNs and increased mortality of beetle larvae (D. abbreviatus) compared to controls in a citrus orchard. In addition, field applications of isolated pregeijerene caused similar results. Quantitative real-time PCR revealed that pregeijerene increased pest mortality by attracting four species of naturally occurring EPNs in the field. Finally, we tested the generality of this root-zone signal by application of pregeijerene in blueberry fields; mortality of larvae (Galleria mellonella and Anomala orientalis) again increased by attracting naturally occurring populations of an EPN. Thus, this specific belowground signal attracts natural enemies of widespread root pests in distinct agricultural systems and may have broad potential in biological control of root pests. Citation: Ali JG, Alborn HT, Campos-Herrera R, Kaplan F, Duncan LW, et al. (2012) Subterranean, Herbivore-Induced Plant Volatile Increases Biological Control Activity of Multiple Beneficial Nematode Species in Distinct Habitats. PLoS ONE 7(6): e38146. doi:10.1371/journal.pone.0038146 Editor: Martin Heil, Centro de Investigacio´ n y de Estudios Avanzados, Mexico Received March 8, 2012; Accepted April 30, 2012; Published June 27, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This research was partially supported by grants from the USDA-CSREES, Citrus Research and Development Foundation and start up funding provided by the University of Florida to LLS. RCH was supported by the Seventh Framework Programme of the European Union (FP7-PEOLE-2009-10F-252980). Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: stelinski@ufl.edu ¤ Current address: Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York, United States of America Introduction Natural enemies of herbivorous pests use flexible foraging strategies that often incorporate environmental cues emitted by the herbivore’s host plant. While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well- documented aboveground [1–5], new evidence from several systems, including strawberry, maize and, most recently, citrus, indicates that induced root volatiles may protect plants by attracting entomopathogenic nematodes (EPNs) [6–12]. However, to date, only one root-induced attractant has been described and shown to enhance the effectiveness of EPNs in the field: (E)-b- caryophyllene from the roots of maize (Zea Mays L.) [11,13]. The disparity in the number of aboveground investigations versus analogous belowground research on indirect defense is largely due to technical limitations rather than a lack of ecological or agricultural relevance [9,14]. No previous studies have detected a belowground herbivore-induced volatile from intact plants in the field or measured the effectiveness of belowground attractants for recruiting populations of naturally occurring EPNs in the soil. Depending on the specificity of interactions, the identification and manipulation of a root signal in the field could well enhance biological control of diverse root pests in agroecosystems. Larvae of the weevil Diaprepes abbreviatus (L.), introduced into Florida in 1964 [15], feed on the roots of more than 290 plant species including citrus, sugarcane, potatoes, strawberries, sweet potatoes, papaya, and non-cultivated wild plants [16]. Over the past 40 years, the weevil has significantly contributed to the damage and spread of disease in agricultural plants [17]. Because pesticides are expensive, environmentally hazardous and often ineffective [18,19], currently the most effective alternative method of root-pest control is the application of EPNs from the genera Heterorhabditis and Steinernema [20]. EPNs are obligate parasites that kill their host with the aid of a symbiotic bacterium [21,22]. Over its 20 years of use, the efficacy of mass release of EPNs as a biopesticide for D. abbreviatus has been reported as varying and PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38146