Insect culture
A stock culture of A. fabae, originated from field collection of infesting broad bean (Vicia faba L.) plants in the experimental area of Ondokuz Mayis University, Turkey, during 2017 was established. The pest was reared in 30 × 20 × 40 cm cages at 22 ± 1 °C and 16 h photoperiod for several generations (Mohammed 2018).
Fungal cultures
The fungal isolates were obtained from the stock cultures at the Mycology Laboratory, Department of Plant Protection, Faculty of Agriculture, Ondokuz Mayis University, Samsun, Turkey. A total of six isolates of two EPF, i.e., L. muscarium (five isolates) and S. lamellicola (one isolate) were used for bioassays. The fungal isolates used in the present study were identified by Dr. Richard A. Humber, an insect mycologist (USDA-ARS). The isolates were cultured on potato dextrose agar (PDA; Merck, Darmstadt, Germany) for 5–7 days at 25 ± 1 °C. The fungi were stored at 4 °C on PDA dishes until the start of the bioassay experiments.
Conidial germination assessment
The conidium viability of the fungal isolates (TR-01, TR-04, TR-05, TR-07, TR-08, and TR-10) was determined following Saruhan et al. (2015). The conidial suspension was adjusted to 104 conidia/ml, and 100 μl was sprayed on Petri dishes (6 cm diameter) containing PDA. The dishes were then sealed by a parafilm (American National CanTM) and incubated at 25 ± 1 °C. The presence of germinating and non-germinating conidia was counted using an Olympus CX-31 compound microscope (Olympus America Inc., Lake Success, NY) at × 400 magnification after 24 h of incubation. Conidia were regarded as germinated when they produced a germ tube having at least half of the conidial length. The germination ratios of the fungi were determined by examining a minimum of 400 conidia from each of the replicate dishes.
Commercial products
One commercial bioinsecticide, V. lecanii (Nibortem SL, 250 ml/100 l of water), and a synthetic insecticide, Imidacloprid (Conmirid SC 350, 20 ml/100 l of water), were used in the study as a positive control.
Inoculum of isolates of EPF
The six fungal isolates belonging to L. muscarium and S. lamellicola were cultured on PDA at 25 ± 1 °C for 14 days before the initiation of the experiment. Conidial suspensions of the isolates were initially prepared in Tween 20 (0.02% in sterile distilled water) and then filtered through four layers of sterile cheesecloth to remove mycelium and agar pieces. The conidial suspensions were then vortexed for 3 min for homogenization. The concentration of conidial suspension was determined by a Neubauer hemocytometer and adjusted at 1 × 104, 1 × 105, and 1 × 106 conidia/ml.
Experimental design
Conidial suspensions of L. muscarium (five isolates: TR-04, TR-05, TR-07, TR-08, and TR-10) and S. lamellicola (TR-01), V. lecanii, and Imidacloprid were applied on fresh broad bean leaves, obtained from 3-week-old plants grown in pots, containing ten A. fabae (third nymphal instar) placed in Petri dishes (9 cm diameter) with sterile distilled water-soaked blotters. For eight treatments, a 2-ml solution was sprayed by a Potter spray tower (Burkard, Rickmansworth, Hertz, UK) on the nymphs of A. fabae. The Petri dishes were loosely capped to prevent the escape of insects. The same number of nymphs was used for control, where only sterile distilled water containing 0.02% Tween 20 was sprayed. All dishes were incubated at 25 ± 1 °C in 16 h light/8 h dark cycle and in 70 ± 5% RH for 7 days and inspected daily. Dead nymphs were counted, using a Leica EZ4 stereo dissecting scope at × 40–70 magnification, and the mortality rate was calculated per Petri dish. The experiment was repeated twice, with four replicates per treatment.
Measurement of mycelial growth and sporulation
Mycelial growth and sporulation of the isolates belonging to L. muscarium (TR-08) and S. lamellicola (TR-01) were assessed according to Cheng et al. (2016) with slight modifications. Mycelial disks (4 mm in diameter) from 10-day-old fungal cultures were placed in the centers of the Petri dishes (9 cm) containing PDA. Then, the dishes were sealed by a parafilm and incubated at 25 ± 1 °C. Mycelial growth was measured daily at two perpendicular colony diameters up to the point of nearly covering the Petri dishes, and their initial day of sporulation was recorded. At the end of the experiment, three agar pieces of 1 cm2/fungus were cut from the Petri dishes in which fungal growth occurred, using a sterile scalpel and put into 50-ml sterile polypropylene tubes. The conidia produced on each of the PDA pieces were shaken and dispersed in 20 ml of 0.02% Tween 20 solution. Then, the conidia were counted under Olympus CX-31 compound microscope using a Neubauer hemocytometer, and the spore amount per unit area was calculated. The experiment had three replicates/isolate repeated at different times.
Statistical analysis
Mortality data was corrected using Abbott’s formula (Abbott 1925). Serial time-mortality data from bioassays were analyzed by probit analysis using SPSS software (SPSS, version 21) to calculate the lethal times, 50% (LT50) and 90% (LT90). Mortality rates of A. fabae treated with EPF, mycelial growth rate and sporulation of six EPF isolates were compared by one-way analysis of variance (ANOVA), followed by Tukey student size post-hoc test where ANOVA indicated significance (P < 0.05).