Conidiospores production of B. bassiana
An isolate of the fungus B. bassiana isolated from a naturally infected mole cricket (Gryllotalpa gryllotalpa L.) (El Husseini et al. 2008) was cultured on potato dextrose agar (PDA) medium poured into sterilized Petri-dishes (12 cm in diameter). Inoculated Petri-dishes with a spore suspension of B. bassiana were incubated for 15 days at 25 °C. The produced areal conidiospores were harvested from the Petri-dishes by scraping with a spatula and suspended in distilled sterilized water with 0.02% Tween 80 as a wetting agent in the primary stock suspension, following Rombach et al. (1987). The spore count was determined by using a Neubauer Hemocytometer, and the stock was kept in the refrigerator till needed.
Larvae and adults of RPW
Larvae of the RPW were collected from three highly infested date palm trees that suddenly fell on the ground at the Experiment Station at the Faculty of Agriculture, Giza, Egypt, in 2017. A large number of adult weevils were present inside the fallen palm trees associated with many larvae in different instars and cocoons having pupae inside. Adult weevils and larvae were collected by hand and transferred in metal boxes to the laboratory at the Centre of Biological Control, Faculty of Agriculture, Cairo University, Giza, Egypt. Due to the great difference in size and age of the collected larvae, the selected ones were categorized in young (Y) (3rd instar L3) and older (O) as full-grown larvae (7th instar L7). Cut tissue pieces from the inside of the same fallen trees served as a food for both larvae and adults in the laboratory during the experimental period.
Efficacy of B. bassiana versus larvae of RPW
Six concentrations of (6 × 102, 6 × 103, 6 × 104, 6 × 105, 6 × 106, and 2 × 107 spores/ml) were prepared in distilled water from the stock suspension (containing Tween 80) by successive dilutions. A fine perfume atomizer was used to spray the tested spore concentrations directly onto larvae of the two larval instars (L3 and L7). The larvae were treated in five replicates per concentration, each of 10 larvae; i.e., 300 young larvae (L3) and 300 older larvae (L7). After the dryness of the sprayed spore suspension, the treated larvae were transferred, using soft forceps into metal boxes (10 × 15 × 30 cm) with perforated metal cover and provided with cut tissues from date palm trees as food. A control was set of untreated larvae for each instar, i.e., 50 larvae for each and sprayed only with water containing 0.02% Tween 80. The boxes were kept under laboratory conditions of 25 °C and 50–60% RH. Inspection for larval mortality occurred daily for 20 days, and the larvae were provided with food. To prove death by B. bassiana, dead larvae were surface-sterilized for 2–3 s in formaldehyde and rinsed with sterilized distilled water under aseptic conditions. Thereafter, they were placed in sterilized Petri-dishes furnished with wet sterilized filter paper (Merghem, 2011). The Petri-dishes were kept in self-clip closing polyethylene bags to maintain relatively high humidity and kept at room temperature at 25 °C and 50–60% R.H. to allow the development of the fungus.
Efficacy of B. bassiana versus adults of RPW
Five concentrations of (6 × 103, 6 × 104, 6 × 105, 6 × 106, and 2 × 107 spores/ml) were tested against the adults. The conidiospore suspensions were applied directly on the adult weevils in Petri-dishes as mentioned earlier. After the dryness of the sprayed suspension, the weevils were transferred into metal boxes (the same as in case of the larvae) and inspected daily among a period of 10 days to supply with food if needed and to record the mortality. The dead adult weevils were treated as that of the dead larvae to prove death by B. bassiana.