Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves Raquel Campos-Herrera a,b,⇑ , Fahiem E. El-Borai a,c , Robin J. Stuart a , James H. Graham a , Larry W. Duncan a a Entomology and Nematology Department, Citrus Research and Education Center, University of Florida, IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA b Departamento de Contaminación Ambiental, Instituto de Ciencias Agrarias (ICA), CSIC, Serrano 115 Dpdo, Madrid 28006, Spain c Plant Protection Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt article info Article history: Received 23 February 2011 Accepted 14 June 2011 Available online 23 June 2011 Keywords: Heterorhabditis Paenibacillus Quantitative real-time PCR Soil food web Steinernema abstract Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organ- isms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Pae- nibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan Ò probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commer- cially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paeni- bacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bac- terium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research val- idate several new tools for studying the ecology of EPNs and their phoretic bacteria. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Entomopathogenic nematodes (EPNs) in the families Heteror- habditidae and Steinernematidae are promising for use as non- chemical insect pest control agents for many perennial and annual crops (Kaya et al., 2006). Although EPNs are considered obligate parasites of a large number of arthropod species, they can scavenge insect cadavers under certain circumstances (Pu ˚z ˇa and Mrác ˇek, 2010; San-Blas and Gowen, 2008). EPNs are vectors of enteric bac- teria (Xenorhabdus for steinernematids and Photorhabdus for het- erorhabditids) that act synergistically with the nematodes to kill the host and then share the tissues for development and reproduc- tion (Boemare, 2002). EPNs are widely distributed in soils throughout the world (Adams et al., 2006; Hominick, 2002). Many commercial products are available that employ EPNs for pest control, especially in crops under organic production, or systems where the use of chemical products is undesirable (Kaya et al., 2006). Steinernema carpocap- sae, Steinernema feltiae, Steinernema glaseri, Steinernema scapterisci, Heterorhabditis bacteriophora and Heterorhabditis indica are among the many species available in products used nearly worldwide (Kaya et al., 2006). Moreover, several species such as S. carpocapsae, S. feltiae and H. bacteriophora are so broadly distributed across different habitats that they have been described as ubiquitous (Hominick, 2002). Nevertheless, EPN spatial patterns, population density, and insecticidal efficacy vary by soil characteristics, agri- cultural management (Campos-Herrera et al., 2008; Kaspi et al., 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.06.005 ⇑ Corresponding author at: Citrus Research and Education Center (CREC), University of Florida (UF), 700 Experiment Station Road, Lake Alfred, FL 33850, USA. Fax: +1 863 956 4631. E-mail address: r.camposherrera@ufl.edu (R. Campos-Herrera). Journal of Invertebrate Pathology 108 (2011) 30–39 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip