Molecular Identification of Six Steinernema Isolates and Characterization of their Internal Transcribed Spacers Regions

Abstract

Molecular identification and genotyping of entomopathogenic nematodes (EPNs) are prerequisites for their proper classification, biodiversity studies, and their potential use in biological control programs. In Palestine, although several isolates of EPNs have been collected, phenotypically characterized, and assessed for their tolerance to cold and heat stresses, the molecular identification and genotyping of most of these isolates have not been accomplished yet. In this study, genomic DNA was isolated from all nematode stages of six Steinernema isolates collected in several areas of historical Palestine. The Internal Transcribed Spacers 1 and 2 (ITS1 and ITS2) as well as the 5.8S regions of the 26S rDNA were amplified and sequenced. The obtained sequences were aligned to identify, to explore sequence variations and construct phylogenetic relationships among the isolates. Three of the isolates were identified as S. feltiae while the other three as S. carpocapsae. At the interspecies level, the 5.8S sequences were more conserved than the ITS sequences which varied in structure and length. Moreover, the ITS2 sequences contained more variable informative sites than those of ITS1. The aligned sequences of the six isolates were analyzed using the neighbor-joining method for two sets of sequences; ITS1-ITS2, and ITS1-5.8S - ITS2. In both cases, similar clustering profiles were produced with slight differences in the branch length of each cluster. These data showed that the association of the different rDNA regions of these Steinernema isolates with different evolutionary rates is solely at the species level but not among different isolates of the same species.