Sampling, isolation, and culturing of the nematode
Soil sampling and trapping the samples with the last instar larvae of the greater wax moth, Galleria mellonella (L.), was carried out according to Woodring and Kaya (1988) method. The samples were collected from Moghan region, Ardabil province, Iran, during 2015 and 2016. Infective juveniles of the nematodes cultured in the last instar larvae of G. mellonella were stored in 40-ml distilled water at 5 °C.
Morphological and morphometric identification of the nematode
Males and females, collected from 5-day infected Galleria cadavers, were dissected in distilled water. Infective juveniles (IJs) were collected after emergence from Galleria cadavers in White traps (Woodring and Kaya 1988). The nematodes were killed and processed to anhydrous glycerin by a slow evaporation method (Woodring and Kaya 1988), then mounted on microscopic slides. Morphological and morphometric measurements were recorded. The morphometric measurements included the following: L = body length, W = greatest body width, EP = distance from anterior end to excretory pore, NR = distance from anterior end to nerve ring, ES = esophagus length, a = L/W, b = L/ES, c = L/T (T = tail length), D% = EP/ES × 100, E% = EP/T × 100, ABW = anal body width, SL = spicule length, GL = gubernaculum length, IJ = infective juvenile, ratio SW = spicule length/ABW, ratio GS = gubernaculum length/spicule, MUC = mucron, A = absent, P = present.
Molecular identification of the nematode: DNA extraction, amplification, and sequencing
DNA was extracted from 10 IJs, using by modified method of Hominick et al. (1997). Briefly, the IJs were crashed in 8-μl deionized distilled (dd) H2O in a sterilized 0.5-ml microcentrifuge tube. then 12-μl lysis buffer was added, and the mixture was homogenized for 1 min. After adding 2 μl proteinase K, the tube was frozen at − 20 °C for 2 h. The tube was incubated at 67 °C for 1 h, followed by a 97 °C incubation for 10 min to digest the proteins and inactivate proteinase K. Subsequently, the tube was centrifuged at 4000 rmp for 2 min. The supernatant containing the DNA was collected and used in PCR amplification. The ITS region from of the ribosomal DNA was amplified in a sterile 0.5-ml tube. The following were used to assemble the reaction: 15 μl of Master Mix, 10 μl of dd H2O, 1 μl of 10 pM forward primer, 1 μl of 10 pM reverse primer, and 3 μl DNA extract. The primers were TW81:5′-GTTTCCGTAGGTGAACCTGC-3′ (forward) and AB28:5′-ATATGCTTAAGTTCAGCGGGT-3′ (reverse). PCR reactions were conducted in a Thermocycler. The PCR cycles were as follows (Hominick et al., 1997): 1 cycle at 95 °C for 4 min followed by 33 cycles at 95 °C for 30 s, 57 °C for 40 s, and 72 °C for 30 s. The last step was 72 °C for 5 min. PCR product was purified and sequenced by Bioneer Corporation, Korea. The obtained sequence was deposited at the NCBI (National Center for Biotechnology Information) database with the accession number of MF187616.1. Nucleotide row data was edited using MEGA X software (Kumar et al., 2018), and homologous sequences were involved in analysis using Blast software. Sequences were aligned, using Clustal W. Bootstrap analysis. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei 1993). The phylogenetic tree was constructed by the maximum likelihood method, using MEGA 6.0 software, and Steinernema feltiae was used as out-group.
Insects
Galleria mellonella
The greater wax moth, G. mellonella, was reared in plastic boxes with ventilated lids at 26 ± 2 °C, 50 ± 5% RH, and 16:8 (L:D) photoperiod and on an artificial diet composed of 1200-g wheat flour (44.1%), 600-g honey (22.1%), 500 ml of 99% glycerol (18.4%), 300-g yeast (11%), and 120-g bee wax (4.4%).
Agrotis segetum
Last instar larvae of A. segetum were collected from tomato fields in Ardebil Agricultural Research Station in Moghan region and transferred to the laboratory of Plant Protection Research Department. The larvae were reared on corn seedlings. Prepupae were placed in the egg lying dishes described by Sherlock (1979). The eggs were collected, and the developed last instar larvae were treated based on Sherlock (1979) method, with exception of using fresh corn seedlings as feeding source for the larvae. Rearing conditions were 25 ± 1 °C and of 70 ± 10% RH and photoperiod 16:8 (L:D). In 24-h time intervals, last instar larvae were collected from rearing dishes and were used in the bioassay experiments.
Nematode
Infective juveniles of S. carpocapsae IRMoghan were cultured on the last instar larvae of G. mellonella (Woodring and Kaya, 1988). The IJs were stored in distilled water at 5 °C and used in all experiments within 30 days of emerging from the host. Before starting the experiments, nematodes were kept at 25 °C for 20–30 min.
Bioassay
Last instar larvae of A. segetum were used in the bioassay experiments. The experiments were conducted in plastic cups (9 cm height and 7.5 cm diameter), filled with 250-g autoclaved moist sandy soil (85 sand, 10 silt and 5 clay and 10% moisture (w/w)). Based on preliminary experiments, a range of 10–100 IJs per insect (i.e., 10, 25, 50, 75, and 100 IJs per larva) was used. IJs were added in certain concentrations in 0.5 ml of water to the surface of the soil, separately. Finally, one larva was placed on the soil, and the cups were covered by ventilated lids to avoid desiccation. Control cups received 0.5-ml distilled water without nematodes. A piece of stem of fresh corn seedlings was used as feeding source which was renewed daily. Fifteen cups were used for each nematode concentration and the control. The dead or alive insects were counted after 7 days. The experiment was replicated 3 times. All dead insects were collected and dissected to ensure the presence of nematodes inside the cadavers.
Statistical analysis
LC20, LC50, and LC80 values were obtained by Probit analysis, using SAS software (SAS Institute 2004). Analysis of variance was done, and the means were evaluated by Duncan’s multiple-range test (SAS Institute 2004). Lethal experimental data was transformed into square root of (x + 1) where needed, before analysis.