Long-term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real-time PCR assays

Quantitative real-time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species-complex in the Steinernema glaseri- group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species-specific primers and TaqMan (R) probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8-ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000-01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baiting method for detecting the EPN species composition in population mixtures. Moreover, the spatial patterns of EPN in this orchard were remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.