Insect culture
Adults of T. castaneum were obtained from the culture that reared in the laboratory on wheat flour at 28 ± 2 °C and 65–75% R.H. The cultures were kept at the stored grain management cell, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. All adult insects used in these tests were 6–7 days old.
Grains
Untreated, uncontaminated, and infestation-free grains of wheat (var. Galaxy) were used in the tests. Wheat grains were obtained from the 2017 harvest. The grain moisture content, as determined by the Grain Moisture Tester RICETER f505 (Kett Electrical Laboratory, Tokyo, Japan), was 11.7%. The grains were held at an ambient condition for 7 days to equilibrate to the desired RH before use in the experiments.
Fungal formulations
The strain of B. bassiana isolate used in this study was obtained from dead cadavers of rice leaf folder, Cnaphalocrocis medinalis (Guenée) (Rizwan et al. 2019). The fungus was then sub-cultured on Potato Dextrose Agar (PDA) plates for bulk generation of the fungal conidia. For bulk conidial production, plates were incubated for 14 days at 20 ± 1 °C and 16 h light per day. Then, the conidia were collected by scraping the conidial layers developed on the plate surface by a sterilized scalpel. The collected conidia were mixed into 100 ml sterile distilled water and filtered through muslin cloth. The fungal conidia and dust carrier were mixed at 1:4 ratio for formulation preparation (Kavallieratos et al. 2006). The conidia collected from strain were comprehensively mixed with the inert carrier ash in screw-capped bottles. Fungal conidia concentration was determined in the conidial suspension, using a hemocytometer. Two concentrations of B. bassiana were prepared, containing 1 × 106 and 1 × 108 conidia kg−1 of wheat grains (Michalaki et al. 2006 and Kavallieratos et al. 2006).
DE formulation
The DE formulation Diafil 610, used in this study, was manufactured by Celite Corporation (Lompoc, CA, USA). It contains 89% amorphous SiO2, 4.0% aluminum oxide (Al2O3), 1.7% iron oxide (Fe2O3), 1.4% CaO, < 1% MgO and K2O, and 3% moisture. This DE was used at concentrations of 200 and 400 ppm (equivalent to 0.20 and 0.40 g/kg of wheat grains, respectively).
Grain treatment
Eight concentrations of EPF, DE, and their combinations were tested, i.e., the lowest concentration (1 × 106 conidia kg−1 of wheat) of the fungus alone, the highest concentration (1 × 108 conidia kg−1 of wheat) of the fungus alone, the lowest concentration (200 ppm) of DE, the highest concentration (400 ppm) of DE alone, and the combinations of low fungal concentration (1 × 106 conidia kg−1 of wheat) + low DE concentration (200 ppm), low fungal concentration (1 × 106 conidia kg−1 of wheat) + high DE concentration (400 ppm), high fungal concentration (1 × 108 conidia kg−1 of wheat) + low DE concentration (200 ppm), and high fungal concentration (1 × 108 conidia kg−1 of wheat) + highest DE concentration (400 ppm). For each grain treatment replication, lots of 1000 g wheat grains were organized and the particular quantity of fungus (1 g for each concentration corresponding to 1 × 106 and 1 × 108 conidia kg−1 of wheat) and DE (0.20 and 0.40 g) were added. These lots were introduced in plastic jars (24 × 14 × 14 cm), and the jars were shaken manually for approximately 5 min to attain an equal dispersal of the dust on the whole grain mass. There was an additional untreated lot which served as control. All jars were kept in a laboratory at 28 ± 2 °C and 65 ± 5% RH during the whole experimental period.
Bioassays
The post treatment efficacy for F1 was observed for the time period of 2 months. Wheat batches of 1 kg were used to apply different treatments, viz., B. bassiana alone, DE alone, and their combinations. Nine samples (eight treatments and one control), each of 50 g, of wheat were used in this experiment. Each sample was placed in a cylindrical plastic jar (24 × 14 × 14 cm) with a top covered with muslin cloth for aeration, and 40 adults of T. castaneum were introduced into each jar. These jars were placed in a laboratory at room temperature and 65 ± 5% RH (Kavallieratos et al. 2006). The desired RH in the laboratory was maintained by using a humidifier. The number of dead adults was counted after (7, 14, and 21 days) in treated and untreated jars. The adults were classified as dead (unable to move even with stimulus) and alive (moving/or showing signs of activity). Thus, the data for mortality was recorded. The adults were removed from the jars after final observation. The mycosis data were recorded from the dead cadavers of T. castaneum that were collected from each treatment upon mortality assay termination. These cadavers were washed (two to three times) by 0.05% sodium hypochlorite solution, followed by three to four washings in distilled water, and then placed on PDA plates. These were incubated at 25 ± 2 °C and 75 ± 5% RH for 1 week and then observed under a microscope for white fungal growth.
Progeny production counts
The dead and alive adults were removed from the jars, and these jars were placed undisturbed for another 60 days to ascertain the progeny emergence. After the completion of 60 days, the emerged adults were counted in each jar.
Data analysis
The counts for mortality rates were converted into percent and then analyzed using Statistix 8.1 software; however, the control mortality was very low and was not included in the analysis. Data were analyzed in three factorial CRD design, and the means were separated using the Bonferroni test at P = 0.05.