Date of this Version
Florida Entomologist 92(4) December 2009, pp. 661-663.
Entomopathogenic nematodes (EPN) belong to the families Steinernematidae and Heterorhabditidae. They infect a wide range of insect hosts and are used to control numerous species of soil borne insect pests (Grewal 2002). We identified EPN genera based on the color of cadavers of the wax moth, Galleria mellonella L., in which Heterorhabditis and Steinernema, produce red and tan colors, respectively, according to Woodring & Kaya (1988). We obtained an isolate of an EPN from sandy loam soil with the Galleria technique (Bedding & Akhurst, 1975) from grasslands in the Campus Tecomán of the Universidad de Colima, and we designated it as JMO94. In 2008, the morphometry and molecular characterization of JMO94 was conducted at the University of Florida Gainesville, Department of Entomology and Nematology under the supervision of Dr. K. B. Nguyen, and our JMO94 isolate was identified as Steinernema diaprepesi by morphological, morphometrical, and molecular results. Our strain is now labeled as S. diaprepesi Colimense strain, and represents a new record of occurrence of S. diaprepesi outside of the United States. DNA extraction, PCR amplification, sequencing, multiple alignments and other processes were conducted by methodology reported by Nguyen et al. (2001, 2004, 2006). PCR amplification was conducted by methodology reported by Nguyen et al. (2004) with the following exceptions: for ITS regions, the 2 internal primers for ITS regions suggested by Nguyen et al. (2001): KN58 = 5’-GTATGTTTGGTTGAAGGTC- 3’ and KNRV = 5’-CACGCTCATACAACTGCTC- 3’, were used; and for region D2/D3 regions, the primers D2F, 5’-CCTTAGTAACGGCGAGTGAAA- 3’ (forward) and 536, 5’-CAGCTATCCTGAGGAAAC- 3’ (reverse), were used as external primers to amplify the D2/D3 regions; primers 502, 5’-CAAGTACCGTGAGGGAAAGTTGC- 3’ (forward) and 503, 5’-CCTTGGTCCGTGTTTCAAGACG- 3’ (reverse), were used as internal primers for sequencing. Molecular phylogenetic relationships were obtained by maximum parsimony (MP) with PAUP, 4.0b8 (Swofford 2002). For phylogenetic analysis of the ITS regions, Steinernema intermedium was treated as the outgroup taxon for resolving relationships among the rest of Steinernema species (Nguyen et al. 2001). For D2/D3 regions, Panagrellus redivivus was used as the outgroup taxon (Stock et al. 2001). Branch support was estimated by bootstrap analysis (1000 replicates) based on the same parameters as the original search.