Rearing of Spodoptera littoralis larvae
Spodoptera littoralis larvae were reared on artificial diet under the laboratory conditions of 25 ± 1 °C and 65 ± 5% (Güney et al. 2019). Emerged adults were fed on honey solution in impregnated cotton in plastic boxes (36 × 23 cm). Egg masses were collected on paper band regularly and transferred into new storage boxes including artificial diet for maintaining the colony and for experimental uses. The second-instar larvae (L2) were used for testing the pathogenicity of the fungal isolates in the experiments.
Fungal isolates
In the experiment, 64 EPF isolates were isolated from soil samples taken from Ordu Province in Turkey in 2019–20 using the Galleria bait method (Zimmermann 1986). DNA extraction of isolated fungi was performed for the identification of the isolates. Then, the polymerase chain reaction (PCR) was carried out to genomic DNA amplify, using ITS4/ITS5 primers. Finally, the obtained PCR products were subjected to sequence analysis. As a result of the sequence analysis, it was determined that the isolates were 23 Beauveria bassiana, 11 Metarhizium brunneum, 8 M. anisopliae, 6 M. robertsii, 4 Purpureocillium lilacinum, 4 Clonostachys rogersoniana, 3 Fusarium solani, 1 Clonostachys rossmaniae, 1 Aspergillus flavus, 1 Cordyceps cicadae, 1 C. fumosorosea and 1 F. oxysporum isolates. All isolates were grown on potato dextrose agar (PDA) medium in incubator at 25 ± 1 °C for 15–30 days.
Preparation of fungal inoculum
To produce inoculum, the fungi were subcultured to PDA plates by conidial transfer. Fungal spores were harvested by scraping using scalpel to falcon tubes after getting sporulation by adding 10 ml of 0.02% Tween 80 solution. The conidial suspension was mixed for 1–2 min and filtered through four layers of cheesecloths to eliminate hyphal fragments. The suspension was diluted to a concentration of 1 × 108 conidia/ml, using a hemocytometer. The isolates displaying the highest mortality rates as a result of single-concentration response tests were adjusted to 1 × 105, 1 × 106 1 × 107, 1 × 108 and 1 × 109 conidia/ml by dilution with the same technique. All suspensions were stored at + 4 °C to be used within 3 days.
Single-concentration response tests
Second-instar larvae of S. littoralis were placed in petri dishes containing artificial diet and sprayed with 1 × 108 conidia/ml concentrations of all isolates. The larvae in control treatment were sprayed by 0.02% Tween 80 solution. Each treatment had a batch of 10 larvae and replicated six times. Mortality rates of the larvae were recorded daily from the 3rd day up to the 13th day of incubation.
Concentration–response tests
Second-instar larvae of S. littoralis were placed in petri dishes as noted above and sprayed with different spore concentrations (1 × 105, 1 × 106 1 × 107, 1 × 108 and 1 × 109 conidia/ml). Mortality rates of the larvae were recorded daily from the 3rd day up to the 13th day of incubation.
Statistical analysis
Average values of larvae mortality data were subjected to probit analysis for calculating LC50 and LC90. Data were processed by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test for comparison of means using the SPSS (Statistical Package of Social Sciences) software version 22.