Rearing of S. littoralis
A laboratory susceptible strain of the cotton leafworm, S. littoralis was obtained as egg masses from the research division of the cotton leafworm, Plant Protection Research Institute (PPRI), Agricultural Research Center (ARC), Giza, Egypt. It was reared on castor bean leaves ever since free from any insecticidal exposure for more than 10 generations under the laboratory conditions (26 ± 2 °C, 65 ± 10% relative humidity and 8:16 light to dark photoperiod).
Selection and maintenance of fungal cultures
The entomopathogenic fungal isolate, B. bassiana Y-F_ITS1 (accession no. [MK773644.1]) used in this study, was isolated from a soil sample collected from agricultural fields in Dakahlia Governorate, Egypt, using the greater wax moth, Galleria mellonella L. (Fergani and Yehia, 2020). Fungal spores were cultivated at 25.0 ± 1.0 °C on Sabouraud dextrose yeast agar (SDAY) medium for 14 days in darkness. Petri dishes showed good growth and spore production traits were selected. After 15 days, spores were harvested by scraping the Petri dishes off with a sterilized glass slide and collected in vials containing 5 ml of sterilized 0.05% Tween 20 (0.1%, v/v). The suspensions were filtered and then vortexed for 3 min. by shaking. All spore concentrations were assessed by direct count using a hemocytometer chamber to ensure the accuracy of the prepared concentrations. To estimate the median lethal concentration (LC50) and lethal concentration 90% (LC90), a series of dilutions were prepared in sterile distilled water to give the range concentrations of 1 × 106, 1 × 107, 1 × 108, and 1 × 109 spores ml−1 for each bioassay. Spore suspensions were used in all the bioassay studies that were less than 6 h, prepared and were stored on ice.
Pathogenicity of B. bassiana against larval instars (L2:L5) of S. littoralis
Larval instars of S. littoralis (L2–L5) were used in the present experiment. For treatment experiments and control, 30 larvae per replicate were placed in clean glass Petri dishes. Castor leaves were provided to each Petri dish for nutrition. For each tested concentration, 30 larvae were placed on a folded Whatman No. 1 filter paper in a glass funnel, and then 20 ml of the spore suspensions were poured. Sterile distilled water with 0.1% Tween 20 was used for the control treatment. Treated larvae were left to move freely for 1 min to dry and then transferred to the Petri dishes lined with filter paper to remove excess humidity. All treatments and control were incubated at 25 °C. The whole experiment was repeated 3 times. Mortality rates at each treatment were recorded daily until the 5th day post-treatment. Dead larvae were kept separately in Petri dishes lined with sterilized moistened filter paper and fungal growth was recorded daily for mycosis test to confirm mortality due to infection by the tested isolate.
Effect of B. bassiana on adult emergence of S. littoralis
Twenty newly formed pupae were transferred gently into a 25-ml glass beaker for each concentration. Ten milliliters of either fungal spore suspension or 0.1% Tween 20 for control was decanted into the beaker, the immersed pupae were shacked gently for 10 s; then, the suspension was poured and the pupae were transferred to clean plastic pots. All pots were lined with moistened filter paper to maintain high humidity for 24 h. After 24 h, the moistened filter paper was removed. Three replicates were used for each concentration. The percentage of adult emergence was observed and recorded based on the number of pupae that did not develop successfully into viable adults. The emerged adults were also monitored for fungal growth and morphological changes.
Bio-efficacy of B. bassiana against S. littoralis eggs
The concentration-effect of different dilutions of spore suspensions on eggs was assessed. Autoclaved Petri dishes, for each concentration, were used for egg treatment. Each Petri dish was packed with a layer of cotton at the bottom covered with a layer of tissue paper. Two milliliters of sterile distilled water was added to each Petri dish for humidity. One egg patch for each treatment was surface sterilized by 0.5% (v/v) aqueous solution of sodium hypochlorite followed by washing with sterile distilled water. Egg patches were left to dry and then immersed in 2 ml of each tested spore suspension with gentle shaking for 2 min. Excess water was removed, using a sterilized filter paper and the egg patches were transferred to the previously prepared Petri dishes. Average number of eggs in each batch was 250 eggs. Each treatment was replicated 3 times and each egg batch was considered one replicate. The control groups were treated with sterile distilled water containing Tween 20 (0.1%, v/v). The treated and control group were incubated at 25 °C and were examined daily for 7 days. The color changes in eggs were observed daily. Egg hatchability was recorded and the unhatched eggs were considered dead.
S. littoralis eggs and larval mortality rates caused by the tested EPF isolate was corrected according to Abbott’s formula (Abbott 1925). LC50 and LC90 values were estimated according to Finney (1971) using “LdPLine®” software [http://embakr.tripod.com/ldpline/ldpline.htm]. Means were analyzed by one-way analysis of variance (ANOVA). Tukey’s method of multiple comparison was done using the statistical program Minitab 16.0 at P ≤ 0.05 significance level (Minitab Ltd., Coventry, UK). The reduction of adult emergence was calculated according to Khazanie (1979). Reduction % = (C − T/C) × 100, where C = number of emerged adults in control and T = number of emerged adults after treatment.