Rearing of tested insect
The tested insect pest, E. cautella, was collected from infested date fruits and was reared at Date Palm Pests and Diseases Department, Central Laboratory of Date Palm, Agricultural Research Center, Giza, Egypt. Adult insects were reared on semi-dry date fruits Siwi cultivar. The date fruits used in rearing culture were conserved at the freezer for 2 weeks before using them to kill potential contamination with other pests. The 2 stages of E. cautella eggs and larvae were separately evaluated. All insect's cultures and experiments were conducted at 26 ± 2 °C and 65 ± 5% (RH), with 16 h of light and 8 h. of darkness (Abd El-Aziz et al. 2012).
Isolation and identification of fungi
Two isolates of the EPF were isolated at Bio-insecticide Production Unit, Plant Protection Research Institute, Agricultural Research Center (ARC), Giza, Egypt. The first isolate, Beauveria Bassiana (AUMC 9808) was isolated from diseased larvae of the cotton leaf worm Spodoptera littoralis (Boisduval 1833) collected from a sugar beet field and was identified at the Mycological Center, Faculty of Science, Assiut University (Sahar and Moharram 2014). The second isolate, Metarhizium anisopliae was isolated from red palm weevil, Rhynchophorus ferrugineus (Olivier 1790). Eggs were obtained from the Department of Date Palm Pests and Diseases, Central Laboratory for Date Palm, ARC, Giza, Egypt, covered with fungi placed on Czapek-dox agar (CZA) medium and incubated at 27 ± 2 °C for 7–10 days to allow growth of infecting fungus. Pure slant culture of fungus was identified in Plant Pathology Research Institute, ARC, Giza, Egypt.
The isolates were cultured on (CZA) medium with 1% yeast extract plates in several Petri dishes (9 cm in diameter), and were grown for 15 days at 27 ± 2 °C. The conidia were harvested by scraping the surface of 14–15 days old culture gently with inoculation needle. The conidia were suspended in distilled water containing 0.1% Tween-80. The mixture was stirred by a magnetic shaker for 10 min and then 4 concentrations of spore's suspension were prepared from each of the fungal isolates (3 × 105, 3 × 106, 3 × 107 and 3 × 108 spores/ml).
Culture of entomopathogenic bacterium
One strain of Bacillus thuringiensis subsp. Kurstaki (Btk) was obtained from the Bio-insecticide Production Unit, Plant Protection Research Institute ARC, Giza, Egypt. T3 medium which was composed of (tryptone 3.0 g, tryptose 2.0 g, yeast extract 1.5 g, MnCl2 0.005 g, and NaH2PO4. H2O 8.9 g, adjusted PH at 6.8), was prepared and the final volume was made up to 1 L with distilled water. The medium was sterilized at 121 °C for 20 min, and inoculation was occurred with the standard inoculums. The inoculated flask was incubated on a shaker (142 rpm) at 28 °C for 72 h. (Attathom et al. 1995). Four concentrations (2 × 108, 2 × 109, 2 × 1010, and 2 × 1011 spores/ml) were prepared by plate count method.
Efficacy of entomopathogenic fungi on eggs of E. cautella
Fifty eggs of E. cautella (24:48 h. old) were placed each into a small glass jar (200 ml volume), every jar was treated with the 4 concentrations of each fungal concentration (3 × 105, 3 × 106, 3 × 107 and 3 × 108 spores/ml) then filled with about 50 g Siwi date fruits and covered with cloths fixed with rubber bands. The reduction percentage of eggs hatchability was calculated after treatments. Then the calculated LC50 values of each fungus were combined to study their effects on E. cautella eggs. Then the percentage reduction in egg hatching was calculated.
Efficacy of entomopathogenic fungi and bacterium on larvae of E. cautella
B. bassiana, M. anisopliae and B. thuringiensis were sprayed against 30 individuals of 2nd E. cautella larvae that placed into a glass container (200 ml volume). The larval instar was chosen before penetrating the date fruit and feeding on it from inside. Four concentrations of each fungus (3.0 × 105, 3.0 × 106, 3.0 × 107 and 3.0 × 108 spores/ml) and 4 as well of the bacterium B. thuringiensis (2.0 × 108, 2.0 × 109, 2.0 × 1010 and 2.0 × 1011 spores/ml) were tested. Each pathogen was applied by 2 methods (single and mixed) against the date fruit pest E. cautella in stored date fruit under laboratory conditions of 26 ± 2 °C and 65 ± 5% R.H. The experiment was replicated 4 times. The number of dead larvae in each jar was counted on specific dates after 3, 5, 7, and 14 days from the treatments and the percentages of mortality were recorded.
Combination of the three entomopathogens
The LC50 value of the tested fungus and bacterium was calculated, to study their mixing effects. The different combinations B. bassiana + M. anisopliae, B. bassiana + B. thuringiensis, M. anisopliae + B. thuringiensis and B. bassiana + M. anisopliae + B. thuringiensis were evaluated to study the pathogens combination on E. cautella mortality.
Mortality rates of insects were corrected using the Abbott formula (Abbott 1925) compared to the control (untreated). LC50 and LC90 were calculated through the probit analysis as described by Finney (1971). Comparison between the mortality percentages using the LC50 values of the three tested pathogens combinations were analyzed by Proc., ANOVA in SAS (SAS Institute 2006).