S. littoralis larvae
A laboratory strain of S. littoralis was reared for many generations on the semi-synthetic diet of Shorey and Hale (1965) at 25 ± 1 °C and 50–60% RH at the Centre of Biological Control, Faculty of Agriculture, Cairo University, Egypt. The prepared diet was poured before solidifying into trays (20 × 30 cm) in a layer of 2 cm in thickness. A plastic grid of 80 cubic cells (2 × 2 × 3 cm) was pressed on the diet tray. One larva was confined in each cell on the diet and the structure was covered with a plastic plate perforated with four fine openings over each cell for aeration. The diet amount in each cell is quite enough for the larva to complete development and pupate. The third (L3) and fifth (L5) larval instars were used for testing the efficacy of M. anisopliae isolate.
Propagation of M. anisopliae
A local strain of the fungus originally isolated from a naturally infected mole cricket, Gryllotalpa gryllotalpa L. (El-Husseini et al. 2008) was propagated on Czapek’s Dox agar medium. The inoculated plates were incubated for 15 days at 25 °C and 50–60% RH. Thereafter, the conidia were harvested from the surface of the cultures by scraping with a sterile solution of 0.01% Tween-80. The concentration of the resulted stock suspension was estimated, using a hemocytometer and stored in the refrigerator till needed.
Seven concentrations of (2 × 101, 2 × 102, 2 × 103, 2 × 104, 2 × 105, 2 × 106, and 2 × 107 conidiospore/ml) were prepared in a distilled water from the stock spore suspension. For each tested concentration, 4 replicates, each of 25 larvae in the third and fifth instars were treated by direct spray, using a fine perfume atomizer (El-Husseini et al. 2008). Thereafter, the treated larvae were placed in the cells of the diet trays and left to feed on under a perforated cover plate similarly to those in the rearing technique. For the control, the larvae were sprayed with a distilled water containing 0.01% Tween 80 and kept on diet as those of the treatments. The mortality rate was recorded daily for 10 days post-treatment. The dead larvae were kept on moistened filter paper in Petri dishes and checked daily for the Green Muscardine symptoms proving the death by M. anisopliae.
LC50 and LC90 as well as LT50 and LT90 were calculated, using the software “Ldp Line” software (Bakr 2005). Data were processed by analysis of variance (one-way classification ANOVA), followed by a least significant difference, L.S.D. at 5% (CoStat statistical software).