Target insect
Rearing of Agrotis ipsilon
Agrotis ipsilon larvae used in this study were collected from a vegetable field at Giza Governorate, Egypt, latitude 31.01° north and height above the sea level 30 m. The 4th instar larvae were reared singly inside plastic tubes (1.5 cm in diameter, 15 cm in height) or in small groups in plastic jars to avoid cannibalism until the last instar of the larvae developed to the pupal stage. All pupae were transferred into suitable cages, emerging adult moths were fed by on 20% honey solution till mating and laying eggs. All rearing procedures were carried out at 25 ± 1°C and 75 ± 5% RH (Zhang et al. 2019). The newly hatched larvae were transferred into small plastic jars and provided daily with castor bean leaves (Ricinus communis L.) as a source of food.
Rearing of Galleria mellonella
The greater wax moth, G. mellonella (L.), larvae were collected from injured hives and put in jars (2 kg capacity) until the appearance of moths and reared according to the method illustrated by Birah et al. (2008); the media used are (wheat (130 g), wheat bran (130 g), milk powder (130 g), maize flour (97.5 g), yeast powder (97.5 g), wax (26 g), honey (195 ml) and glycerol (195 ml). Related components with different rates were modified the used media later by Huang et al. (2010).
Entomopathogenic nematode strains
Heterorhabditis strains: Heterorhabditis bacteriophora Pionar (Hb88 strain) was obtained from Randy Gaugler, Rutgers University, New Brunswick NJ, USA, and Heterorhabditis strain TAN5 that isolated by Nouh (2021) was collected from the Noubaria El-Bhaira governorate, Egypt, at Egyptian clover (Trifolium alexandrinum). Mass culturing of both nematode strains occurred in vivo using larvae of G. mellonella as a host (Woodring and Kaya 1988).
Effect of temperature
Infection of the EPNs was carried out to the 4th and 6th larval instars and 3-day-old pupae of A. ipsilon in plastic cups (30 cm3) half-filled with moistened sterile sandy soil (agricultural sandy soil were obtained from Nubarya, Beheira Governorate, Egypt (latitude 31.03° north and height above the sea level 9 m) and used in all the laboratory experiments after being sterilized) and then covered with plastic lids. A 100g of castor bean leaves (R. communis) were offered for daily food. Cups were treated with each strain of Heterorhabditis at 5 concentrations 30, 60, 120, 240, and 480 IJs/cm2 of soil surface. Ten larvae and 3-day-old pupae were used/cup/5 replicates/concentration. The experiments were carried out at three temperatures 20, 25, and 30 ± 2°C and 55–60 ± 2% RH. Water content in the soil was 20% of the soil weight.
Effect of soil moisture content
Infection of the EPNs on 4th and 6th larval instars and 3-day-old pupae of A. ipsilon was the same as the previous described method with some changes in soil moisture and temperature. The experiment was conducted at 25 ± 2°C and 55–60 ± 2% RH, and three different water contents in the soil were 10, 15, and 25% of the soil weight. Cups were treated by the two Heterorhabditis strains (TAN5 and Hb88) at five concentrations of 30, 60, 120, 240, and 480 IJs/cm2 of soil surface. Ten of larvae or 3-day-old pupae were used/ cup/ 5 replicates/ concentration, with 100-g castor bean leaves (R. communis) as a means of daily nutrition.
Semi-field experiments
This experiment was carried out in large plastic boxes filled with 1-kg moistened sterile sandy soil. Nematode strains used each strain placed individually at concentrations of 1000, 2000, 4000, and 8000 IJs/cm2 where the soil surface was applied by (100 ml) water and mixed with the sterile sandy soil and placed inside boxes. In this experiment, 25 individuals of the 4th instar larvae or 3-day-old pupae were placed in a large plastic box (30 × 15 × 15 cm), covered with plastic lids with the castor leaves (R. communis) as a means of daily nutrition. Larvae and pupae were used/ plastic box/ 4 replicates/ concentration. The experiment was carried out at the temperature of 25°C ± 2 open air in November inside the biological control Department and water content in the soil at 20% and 55–60% ± 2 RH.
In all experiments, mortality rates of 4th and 6th larval instars or 3-day-old pupae of A. ipsilon were transferred after 4 days of treatment to White traps to make sure that they were infected with nematodes (White 1927). The third-stage juveniles (IJs) were harvested from the water surrounding White’s trap within 10–14 days of emergence from their hosts. The control remediation was carried out utilizing distilled water.
Statistical analysis
Mortality rates were corrected according to Abbott’s formula (1925). Mortality rates of A. ipsilon larvae and pupae by Heterorhabditis strains TAN5 and Hb88 were statistically analyzed by ANOVA one way. T test was calculated between the mortality percentage of treated larval and pupal A. ipsilon (Snedecor and Cochran 1980).