Rearing of X. luteola
The initial population of adult X. luteola was collected from elm trees around Pakdasht county, Tehran province, Iran. The ELB was reared on elm leaves in container cages (5 × 10 × 5 cm) covered with cloth mesh. New plants were provided to the culture when necessary. The cages were maintained under laboratory conditions of 25 ± 1 °C, 75 ± 5% RH and a photoperiod of 14:10 h (L: D). After getting larval instars, main experiments were carried out on the 1st, 2nd, and 3rd larval instars.
Preparation of spore suspension
The entomopathogenic fungi, M. anisopliae and B. bassiana, were collected from agricultural soil. To prepare the spore suspension, the surface of fungi colonies were shaved by a sterile scalpel into sterilized distilled water plus two drops of Tween 80 (0.5%) was added into Falcon tubes. To separate spores from the spore’s chain, they were stirred by a shaker for 1 min, and then a uniform suspension of each pathogen was prepared. Counting of spores and preparation of spore density per unit volume were carried out by an improved Neubauer (Hemocytometer) and optical microscope (× 40). To perform the experiments, concentrations of 105, 106, 107, and 108 conidia/ml from M. anisopliae and B. bassiana pathogens were prepared separately. In order to increase the confidence level of concentrations, the count of spores was repeated 3 times.
Viability test of isolates
To evaluate the viability of fungi, spores’ germination test was done. About 24 h before the bioassay test, dilute suspensions of isolates were prepared in Tween 80 (0.5%) solutions. Then 100 μl of suspension was poured into the PDA medium in Petri dishes and maintained at 25 °C. After 18 h, 3 parts of the Petri dishes were specified, and randomly, 100 spores were selected from each part, and it was counted by optical microscope (× 40). When the germ tube length reached more than one half the diameter of spore, it was considered as a germinated spore (Greenfield et al. 2016). The germination rate, more than 85%, was selected for bioassay. The percentage of spore germination was calculated using the formula of Benslim et al. (2016):
$$ \mathrm{Spore}\ \mathrm{germination}\ \mathrm{percentage}=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{germinated}\ \mathrm{spore}}{\mathrm{Number}\ \mathrm{of}\ \mathrm{total}\ \mathrm{spore}\mathrm{s}}\times 100 $$
Pathogenicity of fungal isolates
Each larval instars of ELB were exposed separately to the different fungal conidial concentrations (105, 106, 107, and 108 conidia/ml) of M. anisopliae and B. bassiana at 4 replicates with control treatment. Sterilized distilled water plus two drops of Tween 80 (0.5%) was used as control treatment. For each replicate from the concentrations, 20 larvae from each of the 1st, 2nd, and 3rd instars of ELB were utilized. To eliminate saprophytes from the body surface of ELB, larvae were immersed in a sodium hypochlorite 1% for 20 s and washed in sterilized distilled water for 20 s. Eventually, to remove excess water, they were placed on a Whatman’s filter paper for 1 min.
Inoculation of ELB larvae with fungal pathogen was made by spraying and immersion methods. To ensure that each insect of each treatment receives the same concentration, all same instar larvae were immersed and/or sprayed simultaneously. A volume of 5 ml suspensions of each concentration was poured into Petri dishes. Then, suspension was got out, and additional suspension was removed using a Whatman’s filter paper. Within each Petri dish, a moist sterile cotton ball was added to provide moisture. In both spray and immersion methods, 5 ml of each concentration of the 2 fungi were utilized separately on the 1st, 2nd, and 3rd larval instars of ELB. The Petri dishes were covered by parafilm and then placed in an incubator at 25 ± 1 °C and 75 ± 5% for 24 h. After 24 h, dead insects and wet cotton were removed. In the following, sterilized new elm leaves were placed at disposal of the larvae, and Petri dishes were covered with a net lid. Sterilized elm leaves were provided daily to the larvae. Petri dishes were maintained in an incubator at controllable conditions of 25 ± 1 °C, 75 ± 5 RH and a photoperiod of 14:10 h (L: D). Eventually, mortality means of both EPF were counted and recorded in both spraying and immersion methods for 10 days at 48-h intervals.
Statistical analysis
The percentage of mortality was corrected using Abbott’s formula (1925). Probit analysis was used to determine the values of lethal concentrations (LC50 and LC90). Arcsine transformation was applied to mortality data and then subjected to one-way analysis of variance (ANOVA). Means were compared using Tukey’s HSD test at P < 0.05 significance level (Institute SPSS 2018). Student’s t test was performed for comparison of both fungi and two methods.