Nematode culture
Infective juveniles of Steinernema carpocapsae (Bakışlı 05), S. feltiae (ES-3), Heterorhabditis bacteriophora (TOK-20), H. bacteriophora (11-KG), obtained from the Plant Protection Department of Tokat Gaziosmanpaşa University, Turkey were used in this study. Infective juveniles of the 4 nematode species were reared, using the last instar larvae of Galleria mellonella (L.) according to the procedures described by Kaya and Stock (1997).
Rearing Galleria mellonella larvae
Galleria mellonella larvae were reared on a special diet containing 890 g of flour, 222 g of dry baker’s yeast, 500 g of glycerin, 500 g of honey, 445 g of milk powder and 125 g of beeswax. Honey and glycerin were heated and then added to flour, bran, milk powder, and yeast mixture (Mohamed and Coppel 1983). G. mellonella eggs were placed on the food medium in 1 L glass jars and kept in an incubator with 16/8 h lighting set at 23–24°.
Rearing of entomopathogenic nematodes
Last instar larvae of G. mellonella was used to rear the EPNs for experiments. Ten larvae were placed into a 6-cm diameter small Petri dishes with lined Whatman paper soaked with distilled water. A suspension of infective juveniles of either nematodes were then applied on the G. mellonella larvae. The lid of the Petri dishes were wrapped by a parafilm and placed in the incubator at 20–23 °C. Larval mortality was controlled frequently. Infective EPN juveniles were obtained from infected G. mellonella larvae using the “White trap” method (White 1927). Obtained larvae were placed in culture flasks and kept in a refrigerator at + 10 °C. In order to prevent the nematodes from losing their activity, the same process was repeated by infecting new G. mellonella larvae every 1–2 months and thus the cultures were renewed in Nematology Laboratory.
Rearing of codling moth 1st instar larvae
Codling moth 1st instar larvae used in experiments were reared at 25 ± 1 °C, 65% R.H. with a 16L: 8D photoperiod conditions in a climate cabinet. Fifty adults were released in 2 L plastic container containing an adult diet of cotton soak with honey and water supply from a piece of sponge inserted into a container. A polyethylene sheet was placed on the bottom for oviposition and wet pieces of clothes for keeping moisture both side of the container. Adults laid eggs on polyethylene sheets. The eggs were hatched after approximately 5–6 days. Same day hatched larvae on polyethylene sheets were transferred by the help of a brush to artificial larval diets in Petri dishes. Artificial diet was bought from Southland Products Incorporated, USA. Codling moth is mass rearing in Entomology Laboratory.
Laboratory bioassays at different concentrations of EPNs
All laboratory studies were carried out under laboratory conditions (25 °C and 65% RH) using a 6-cm-diameter small Petri dish with artifical diet. Ten laboratory-reared first instar larvae of codling moth taken from the stock culture were placed in a new Petri dish in climate chamber. Four nematode concentrations (0, 25, 50, and 100 IJs/larva) were applied directly to the larvae by pipette (Fig. 1). Deltamethrin was used as a positive control. Larval mortality was calculated after 96 h. Laboratory studies were conducted 5 times for each concentrations per EPN species. There were ten of 1st instar larvae in each petri dish. The experiment was repeated 4 times under the same conditions on different dates.
Experiments were conducted at 24 ± 5 °C and 65% ± 5% RH under a 16 h light/8 dark cycle. Dead larvae were placed on a White trap and after a week, EPN larvae were obtained from infected codling moth larvae. Insect cadavers were examined in distilled water under a stereomicroscope.
Statistical analysis
All data were analyzed, using statistical program JMP 7. Statistical analyses were done using one-way ANOVA, followed by LSMeans Student’s t test to record the differences in the effects of different nematode species and concentrations on codling moth.
