Materials
Seven EPN strains of Steinernema glaseri (HBgy and Sgib strains) (Rhabditida: Steinernematidae), S. carpocapsae (HB310 and All strains) (Rhabditida: Steinernematidae), S. longicaudum (CBZB strain) (Rhabditida: Steinernematidae), and Heterorhabditis bacteriophora (HB8 and HB140 strains) (Rhabditida: Heterorhabditidae) were maintained in Pest Bio-control Laboratory, Hebei Agricultural University, China. The nematodes were cultured in the larvae of the greater wax moth, Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae). Harvested IJs were kept in sterilized water at 10 ± 1 °C in the fridge until they were used in the experiments.
The blackened walnuts were collected from Baoding district (114°50′–115°17′E, 38°45′–39°07′N and 50–1006.7 m a.s.l.), which is located in Hebei Province, China. All the black walnuts were placed on a sandy plate. Matures emerged from the fruit and cocooned in the sand. Under natural conditions, the matures overwinter within cocoon as larval stage in the soil. In this study, the mature larvae and cocoon were selected for the experiments. G. mellonella larvae were obtained from the Pest Bio-control Laboratory, Hebei Agricultural University, China. Larvae were fed on artificial diet (22% maize meal, 22% wheat germ, 11% dried milk, 5.5% dry yeast, 17.5% bee wax, 11% honey, and 11% glycerin) ( Mukherjee et al. 2009) and reared in insectary (28 ± 1 °C, RH 80 ± 5%, and a photoperiod of 14L: 10D).
Larvicidal activity of seven EPN strains against A. hetaohei larvae
Bioassays were conducted to compare the infectivity of seven EPN strains against the mature larvae of A. hetaohei. Twenty mature larvae were placed in Petri dish (90-mm in diameter), filled with two filter papers, which was added with 1 ml IJs suspension (1000 IJs/ml). Petri dishes were sealed with para-film and maintained in a climatic chamber of 25 ± 1 °C and RH 80 ± 5%. Controls received water only. The experiments were carried out three times under the same conditions on different dates with 60 larvae distributed in three replicates (each of 20 larvae). Corrected mortality of the larvae was recorded every 24 h for 120 h, respectively. Dead cadavers were dissected to check for nematodes presence or not under the stereomicroscope.
Determination of desiccation tolerance of seven EPN strains
Desiccation tolerance of nematodes was evaluated under the relative humidity of 50% as described by Chen et al (1999). Each Petri dish filled with two filter papers was provided by 1 ml IJs suspension (1000 IJs/ml). The Petri dishes were kept into the incubator at 25 ± 1 °C, RH 50 ± 5% for 7 h. After then, each Petri dish was added 5 ml distilled water and nematode mortality rate was assessed from three subsamples (100 μl per sample) from each Petri dish under a stereo-zoom microscope. Nematodes were considered dead if they did not respond to prodding.
Determination of the optimal EPN strain against cocooned and mature larvae of A. hetaohei
One optimal EPN strain was gained on the basis of the above experimental results. The infectivity of the optimal EPN to the cocooned and mature larvae of A. hetaohei was tested, respectively. The methods and assessment were done as described above. Larval mortality of each treatment was recorded 48 h post-inoculation. The experiments were carried out three times under the same conditions on different dates with 60 larvae distributed in three replicates (each of 20 larvae).
Influence of temperature and nematode concentrations on virulence of EPNs
Virulence of the optimal EPN strain to A. hetaohei was tested at different temperatures of 16, 20, 24, 28 and 30 °C. Petri dish (90-mm) with two layers of filter paper was added with 20 mature larvae and 1 ml nematode suspension (1000 IJs/ml). Petri dishes were sealed with a para-film and maintained in a climatic chamber at above condition, respectively. Larval mortality of each treatment was recorded after 24, 48, 72 and 96 h post-application. The experiments were carried out three times under the same conditions on different dates with 60 larvae distributed in three replicates (each of 20 larvae).
The virulence of the optimal EPN strain to A. hetaohei larvae was assessed at different nematode concentrations. Bioassay was carried out in 24-well micro-plates. Each well with two layers filter papers was added with one mature larvae and different nematode suspension of 5, 10, 20, 50 and 100 IJs per larva, respectively. All the plates were maintained in a humidity controller at 25 ± 1 °C and RH 80 ± 5%. Larval mortality of each treatment was recorded after 24, 48, 72 and 96 h post-application. Each cadaver was dissected under a stereomicroscope to ensure nematode presence. The experiments were carried out three times under the same conditions on different dates with 60 larvae distributed in three replicates (each of 20 larvae).
Data analysis
All experimental data in the virulence assay were subjected to analysis of variance with treatment as fixed effect (ANOVA). The significant differences between the treatments were determined by using Tukey’s test (p < 0.05). All data analyses were performed using SPSS software (SPSS Inc. 2009).