Insect sources
ABB adults were collected from apple orchards in the research area of Tokat Gaziosmanpaşa University, Faculty of Agriculture (N40°20′01′′ E36°28′27′′, 622 m), in Tokat-Central-Büyükyıldız (N40°20′11′′ E36°23′27′′, 606 m) and in Tokat-Central-Akyamaç (N40°20′47′′ E36°29′53′′, 615 m). The beetles were kept in plastic containers, covered with fine mesh for aeration until used in the bioassay.
Fungal isolates
Fifteen EPF isolates were chosen for the current investigation from the fungal culture collection of the Tokat Gaziosmanpasa University, Faculty of Agriculture, Department of Plant Protection in Tokat, Turkey. These isolates were initially isolated from Hypera postica (Gyllenhal 1813) (Coleoptera, Curculionidae) and Gonioctena fornicata (Brüggemann 1873) (Coleoptera, Chrysomelidae) adults gathered from alfalfa fields in Tokat Province, Turkey (Baysal 2017).
Morphological characterization
Fifteen isolates were preliminarily identified using morphological characteristics such as appearance of the fungal infection, colony shape, spore size and spores shape (Humber 1997).
Molecular characterization
DNA isolation and PCR studies
DNA isolation of the EPF isolates was performed according to the “Genomic DNA Purification Kit Thermo Fisher Scientific” method. ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) as the forward primer and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as reverse primers were used for ITS PCR-amplification (White et al. 1990). For PCR, 1 µl template DNA, 2.5 µl 10X Taq Buffer, 0.2 µl dNTP (25 mM), 0.5 µl ITS5 primer (100 pmol), 0.5 µl ITS4 primer (100 pmol), 1.5 µl MgCl2 (25 mM), 0.25 µl Taq polymerase enzyme (Thermo) and 1 µl of Dimethylsulfoxside (DMSO) were mixed in PCR tubes and made up to 25 µl with distilled water and placed in the termocycler (Techne Prime). Thirty-five cycles were performed in thermocycler following the denaturation at 95 °C for 5 min, 95 °C for 1 min, 55 °C for 55 s, 72 °C for 2 min, with a final extension at 72 °C for 10 min (Sevim et al. 2014). The PCR products obtained as a result of PCR were subjected to electrophoresis at 100 V for 1 h in an agarose gel containing 10 mg/ml ethidium bromide, prepared at a rate of 1.2% and visualized under the imaging device (Sevim et al. 2014).
Phylogenetic analysis
For phylogenetic studies, the products obtained at the end of PCR were sent to Atlas Biotechnology (Ankara-Turkey) for the sequencing. The data obtained at the end of the sequencing were analysed with the MEGAX (Kumar et al. 2018) computer program. The data were then compared with reference isolates registered in the National Center for Biotechnology Information (NCBI) gene bank, and molecular identification of the isolates was made.
Inoculum preparation from entomopathogenic fungal isolates
Fungal isolates were cultured on Potato Dextrose Agar (PDA) in Petri dishes. A small part of mycelia was taken from each fungal isolates and inoculated in of PDA and incubated at 25 ± 2 °C with a 16/8 (L/D) photoperiod. At the end of the 21-day incubation period, spores were harvested with 10 ml of sterilized water containing 0.02% Tween 80. The conidial suspensions were filtered through 3 layers of sterile muslin to remove particles and then conidial concentration adjusted to 1 × 106, 1 × 107, 1 × 108 and 1 × 109 conidia ml−1 (Saruhan et al. 2017).
Bioassays
To determine virulence of isolates against ABB adults, primarily a single- concentration trial was conducted at 1 × 107 conidia ml−1. To test the effect of each of the isolates, ABB adults were immersed into 1 × 107 conidia ml−1 suspension of each isolate for 10 s. and transferred into a Petri dish (5 adults per plate) containing fresh apple leaf and flower. The control group was treated with sterile water including 0.02% Tween 80. Mortality of the adults was assessed on the 3rd, 5th, 7th, 9th, 13th, 15th and 17th days of incubation periods. Furthermore, concentration-mortality trials were conducted similar to single-concentration trials. These tests were performed with some isolates that were determined to have a high effect, employing concentrations of 1 × 106, 1 × 108, and 1 × 109 conidia ml−1. A completely randomized block design with 5 replications was used for the experiments and replicated 2 times.
Statistical analysis
Data was analysed with analysis of variance (ANOVA) and the means were compared to Tukey’s multiple comparison test by using the MINITAB Release 16 packet program. To determine the statistical interactions between treatments, MINITAB Release 16 was used with a general linear model. For isolates tested in concentration-mortality trials, LT30, LT50 and LT90 values at a concentration of 1 × 108 conidia ml−1 were calculated using probit analysis.
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