The study was conducted at Entomological Laboratory of University College of Agriculture and Environmental Sciences, The Islamia University of Bahawalpur, Pakistan.
Insect collection and rearing
Wheat aphid species was collected from insecticide-free wheat fields of the university research area and reared on wheat seedlings grown in plastic pots (15 cm diameter, filled with sandy loam soil and organic matter (2:1), under laboratory conditions (24 ± 1 °C, 65 ± 10% RH), covered with aerial jars. The aphids were allowed to produce nymphs for further generation to get homogenous population (free of insecticidal pressure). From the next generation aphids, second and third nymphal instars were collected to be used in bioassay studies.
Preparation of plant extracts
The fresh leaves of neem, Azadirachta indica L. and Eucalyptus, Eucalyptus camaldulensis D. were collected and washed sufficiently by distilled water. After drying under shade, the leaves were grinded to fine powder with an electrical grinder. 10 g of plant powder was placed in conical flasks (250 ml) along with 100 ml of distilled water and placed on heating (60 °C) and shaked with magnetic stirrer (AM4, Velp Scientifica, Italy) for 6 h to dissolve the leaf powder properly. The solid residues were removed using muslin cloths, and plant extracts were then filtered (Whatman No. 1). After evaporation by using rotary evaporator (HB Digital, Heidolph, Germany) at 60 °C under vacuum, the dried plant extracts were brought to constant volume, using a hot air oven (60 °C) (Ali et al. 2017). These extracts were stored under 4 °C till used as a stock solution. Seven percent concentration of neem and eucalyptus leaf extracts was prepared by dilution with distilled water from this stock solution for experimental evaluation.
Preparation of concentration of entomopathogenic fungi
B. bassiana (RACER™) and M. anisopliae (PACER) were procured from Agri Life Ltd., India, which are formulated at CFU count of 108 fungal spores/g. The concentrations of 106 spores/ml of EPF were obtained by dissolving 1 g EPF formulations in 100 ml of water to apply on the aphids (Agri Life Ltd., India).
Bioassay
Ten S. avenae nymphs (second and third instars) were placed into Petri dish (9 cm diameter) and allowed to set on rooted wheat seedlings (roots covered with wet cotton plugs to keep the seedling alive). After they settled, botanicals extract and EPF were applied with the abovementioned concentrations, using a fine hand sprayer machine (Flip & Spray™ Bottles, Thomas Scientific, USA) with two shower shots to properly cover the Petri dish area. The treatments were set as follows:
T1 water (control); T2 (B. bassiana); T3 (M. anisopliae); T4 (neem extracts; T5 (eucalyptus extracts; T6 (B. bassiana + neem extract); T7 (B. bassiana + eucalyptus extract); T8 (M. anisopliae + neem extract); T9 (M. anisopliae + eucalyptus extract). The concentrations of EPF and botanicals were halved in combination treatments as 1:1. The bioassays were performed under the laboratory conditions at (24 ± 1 °C, 65 ± 10% RH). All treatments were triplicated. To evaluate the fecundity of mature aphids, fourth instar nymphs were treated by botanicals and EPF with the same way, and survived adults were shifted into new Petri dishes with fresh wheat seedlings to give F1 ones.
Data analysis
The aphid mortality rate was recorded on a daily basis. The fecundity data was recorded for 10 aphids from each treatment on a daily basis for 5 days after they started to give young ones. Evaluation of the tested materials and techniques was based on the mortality percentage and corrected by Abbot’s formula (Abbott 1925).
$$ \mathrm{Coreected}\%\mathrm{mortality}=\left(1-\frac{\ \mathrm{n}\ \mathrm{in}\ T\ \mathrm{after}\ \mathrm{treatment}}{\mathrm{nin}\ \mathrm{Co}\ \mathrm{after}\ \mathrm{treatment}}\right)\ast 100 $$
Where n = insect population, T = treated, and Co = control.
The mortality and fecundity data were subjected to factorial analysis of variance (ANOVA), using Minitab 16.1 software. The means were compared by applying Tukey’s HSD test at 5% level of significance to evaluate the impact of treatments on mortality and fecundity of treated wheat aphids.