Entomopathogenic nematodes, phorectic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves

10 páginas, ilustraciones y tablas estadísticas.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 organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus 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 probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially 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 Paenibacillus 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 bacterium 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 validate several new tools for studying the ecology of EPNs and their phoretic bacteria.(CGL-2009-14686-C01-00 BOS y (FP7-PEOPLE-2009-IOF-252980Peer reviewe