First record of Steinernema glaseri Steiner, 1929 (Rhabditida: Steinernematidae) from Belgium: a natural pathogen of Hoplia philanthus (Coleoptera: Scarabaeidae)

Nematology , 2005, Vol. 7(6), 953-956 Short communication First record of Steinernema glaseri Steiner, 1929 (Rhabditida: Steinernematidae) from Belgium: a natural pathogen of Hoplia philanthus (Coleoptera: Scarabaeidae) Minshad Ali A NSARI 1 , Lieven W AEYENBERGE 1 and Maurice M OENS 1 , 2 , ∗ Larvae of the scarab beetle Hoplia philanthus Füessly (Coleoptera: Scarabaeidae) are important pest insects of sports turf, lawns, pastures and ornamentals in Belgium (Ansari et al. , 2003b). During a routine field survey of white grubs in Belgian turfgrass, a number of third-instar larvae of H. philanthus were found infected by individ- uals of the genus Steinernema Travassos, 1927 (Rhabdi- tida: Steinernematidae), a genus of entomopathogenic ne- matodes (EPN) increasingly used as biological control of insect pests (Ehlers, 1998). Despite the progress that has been made in the use of EPN, knowledge about their natural host range and the ef- fect of indigenous nematode populations on local insect populations is limited. Several species of EPN have been isolated from scarabs including H. philanthus (Peters, 1996; Ansari et al. , 2003a) and many have been tested for their biocontrol activity against various economically important insect pests including white grubs (Shapiro- Ilan et al. , 2002). Five Steinernema species, namely S. glaseri (Steiner, 1929) Wouts, Mrá ˇ cek, Gerdin & Bed- ding, 1982, S. arenarium (= S. anomali) (Artyukhovsky, 1967) Wouts, Mrá ˇ cek, Gerdin & Bedding, 1982, S. feltiae (Filipjev, 1934) Wouts, Mrá ˇ cek, Gerdin & Bedding, 1982, S. kushidai Mamiya, 1988, and S. scarabaei Stock & Kop- penhöfer, 2003, have been detected in species of this in- sect group (Glaser & Fox, 1930; Kozodoi, 1984; Poinar, 1986; Mamiya, 1988; Stock & Koppenhöfer, 2003). In laboratory and field tests, many of these species have attained levels of insect control comparable to those achieved by chemical pesticides (Georgis & Gaugler, 1991). The recently isolated species S. scarabaei showed an unusually high pathogenicity to several scarab species 1 Agricultural Research Centre, Department of Crop Protection, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium 2 Laboratory of Agrozoology, Department of Crop Protection, Ghent University, Coupure Links 653, 9000 Ghent, Belgium ∗ Corresponding author, email: [email protected] Received: 7 July 2005; revised: 13 September 2005 Accepted for publication: 13 September 2005 Keywords: biological control, entomopathogenic nematode, insect pathogen, molecular identification, scarab beetle. (Koppenhöfer & Fuzy, 2003). We report here the identifi- cation of a Steinernema species which naturally infected third-instar H. philanthus . Infected larvae of H. philanthus were collected in Oc- tober 2004 in a sports field at Eeklo, province of East Flanders, Belgium. They showed the typical yellow colour of insect cadavers infected with steinernematids. The lar- vae were cleaned and placed onto a White (1927) trap to extract infective juveniles (IJ) of EPN. To verify their pathogenicity, IJ were collected upon emergence from the insect cadaver and transferred onto moist filter paper in Petri dishes to which Galleria mellonella L. (Lepidoptera: Pyralidae) larvae were added. After infection, G. mel- lonella larvae were dissected in Ringer’s solution at 3- or 4-day intervals to obtain developmental stages of the nematodes, which can be used for taxonomic identifica- tion. The nematodes were identified according to a selec- tion of morphological and morphometrical criteria sum- marised by Hominick et al. (1997). To confirm the identification, the nematodes isolated from the Galleria cadavers were characterised using molecular techniques. DNA was extracted from single ne- matodes and amplified as described by Waeyenberge et al. (2000). Primers TW81 and AB28 (Joyce et al. , 1994) were used for amplification of the ITS1-5.8S-ITS2 region. After electrophoresis, amplified products were excised from 1% TAE-buffered agarose gels using the Qiaquick Gel Extraction Kit (Qiagen Benelux B.V., Venlo, The Netherlands), cloned into the pGEM-T vector and trans- formed into JM 109 High Efficiency Competent Cells (Promega, Leiden, The Netherlands). Five colonies were isolated using blue/white selection and used for PCR with vector primers. Amplified products were purified using © Koninklijke Brill NV, Leiden, 2005 953 Also available online - www.brill.nl