Additional data and first record of the entomopathogenic nematode Steinernema weiseri from Turkey

Nematology , 2007, Vol. 9(5), 739-741 Short communication Additional data and first record of the entomopathogenic nematode Steinernema weiseri from Turkey Isik O. U NLU 1 , Ralf-Udo E HLERS and Alper S USURLUK 2 , ∗ Entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae are used to control soil-borne insect pests (Grewal et al ., 2005). In Turkey, the first EPN species found was S. feltiae from soil samples near the Black Sea (Ozer et al ., 1995), S. anatoliense (Hazir et al ., 2003) is so far only known from Turkey and Heterorhabditis bacteriophora was recorded by Susurluk et al . (2001) and Kepenekci and Susurluk (2000). The present study reports another species present in Turkey. Eighty soil samples were taken at a depth of 10 cm on a transect over an area of 50 m 2 at the Beytepe campus of Hacettepe University, Turkey, to check for the pres- ence of entomopathogenic nematodes. Soil composition was 31.68% sand, 23.28% silt and 45.04% clay. Soil pH was 7.55. The total salinity was 0.16% and organic con- tent 4.35%. Soil temperature at that depth was 20.9 ◦ C and the relative humidity was 58.3%. Each soil sample was put into a plastic box (13 cm long × 13 cm wide × 5 cm high) containing five last instar larvae of Galleria mellonella L. (Lepidoptera: Galleriidae) and incubated at 25 ± 2 ◦ C for 5 days (Bedding & Akhurst, 1975). To ex- tract nematodes from dead insects the larvae were trans- ferred to water traps. One sample was found to contain entomopathogenic nematodes of the genus Steinernema . This strain (BEY) was identified molecularly using PCR- RFLP of the ITS region (Internal Transcribed Spacer) of the ribosomal DNA. Total genomic DNA was isolated from 2000-3000 infective juveniles (IJ) with a DNeasy ® Kit (Qiagen, Hilden, Germany) following the instructions of the supplier. DNA was stored at − 27 ◦ C. PCR amplifi- cation of the ITS region was carried out with the primers Institute for Phytopathology, Department for Biotechnology and Biocontrol, Christian-Albrechts-University Kiel, Hermann-Rodewald-Str. 9, 24118 Kiel, Germany 1 Present address: Department of Entomology, Louisiana State University, Baton Rouge, LA 70803, USA 2 Present address: Uludag University, Agriculture Faculty, Department of Plant Protection, 16059 Gorukle-Bursa, Turkey * Corresponding author, e-mail: [email protected] Received: 21 December 2006; revised: 10 April 2007 Accepted for publication: 17 April 2007 Keywords: cross-breeding, molecular, new record, pathogenicity. 18S (5 ′ -TTGATTACGTCCCTGCCCTTT-3 ′) and 26S (5 ′ - TTTCACTCGCCGTTACTAAGG-3 ′) as forward and re- verse primers, respectively (Vrain et al ., 1992). Nine dif- ferent enzymes were used: Alu I, Hae III, Hin dIII, Dde I, Hha I, Hin fI, Hpa II, Rsa I and Sau 3AI (Amersham Bio- sciences, Freiburg, Germany). Molecular identification was performed according to Reid and Hominick (1998). RFLP patterns were compared with published informa- tion on described species (Mrá ˇ cek et al ., 2003; Stock & Hunt, 2005). The RFLP analysis obtained with strain BEY gave identical restriction patterns when compared with the RFLP of the type population of S. weiseri and of S. weiseri (UK isolate 541, previously referred to as RFLP type D1) (Fig. 1). To confirm identity, the BEY strain was cross-bred with the following species; S. weiseri from Czech Republic (Mrá ˇ cek et al ., 2003) and S. feltiae (TUR-S3) from Ankara-Turkey (Kepenekci & Susurluk, 2003) according to the method of Iraki et al . (2000). Infected Galleria were washed in sterile Ringer’s solution (NaCl 9 g, KCl 0.42 g, CaCl 2 · 2H 2 O 0.37 g, NaHCO 3 0.2 g and distilled water 1000 ml) and dissected 5 days after exposure. In order to separate adult from juvenile stages, gradient centrifugation was used. The gradient consisted of 30, 20, 15 and 10% (w/w) Ficoll 70000 (Sigma, St Louis, MO, USA) in distilled water. Nematodes, together with Galleria haemolymph, were put on top of the Ficoll gradient and the suspension was centrifuged at 150 g for 2 min. Debris on top of the 20% Ficoll gradient was discarded. The next layer contained mainly fertilised and unfertilised females of the filial generation and on top of the lower layer (10%) males and juvenile © Koninklijke Brill NV, Leiden, 2007 739 Also available online - www.brill.nl/nemy