Mite culture
Tetranychus urticae was reared on bean plants (Phaseolus vulgaris L.) grown in pots placed in a climate room with temperature of 24 ± 1 °C, relative humidity of 65 ± 1, and 14:10 photoperiod.
Identification of fungus
Morphological identification of fungus
Two isolates of EPF were obtained from the body of insect cadaver. The fungus was morphologically identified under a light microscope according to its conidial features in accordance with the literatures (Samson et al. 1988; Goettel and Inglis 1997; Humber 1998). The isolates were developed on PDA (potato dextrose agar) by incubating them at 23±1 °C, including 12 h light (near ultraviolet light) 12 h dark for 10–12 days. The fungus colonies showed very quick growth by covering Petri dishes in 10 days. The colony color was first white, and then changed close to creamy white. The colonies had a white velvety appearance. Both isolates had an aerial miselium, intense wool-like miselium structure. It was observed that the fungus produced too many conidia. The conidia measured 2–2.5-μm diameter. The conidia occured in 3 days in Petri dishes (Fig. 1).
Fungal pathogens and preparation of the conidial suspensions
In this study, B. bassiana isolates were obtained from the fungal collection of the Plant Protection Research Center and Institute. The 2 isolates were obtained from a body of insect cadaver. BGF14 isolate was isolated from Gonioctena fornicata (Brüggmen) (Coleoptera: Chrysomelidae), while BCA32 was from Cicadatra adanai Kartal (Hemiptera: Cicadidae). Fungus cultures were maintained on Sabouraud dextrose agar (SDA) at 24 ± 1 °C for 21 days. Conidial suspensions were filtered through 3 layers of sterile cheesecloth to remove particles. The number of conidia per milliliter was counted using a hemocytometer. At the end of this period, the conidia were collected in sterile distilled water containing 0.02% Tween 80, and the conidial concentration of the stock culture was adjusted to a density of 1×108 conidia/ml.
Molecular identification of fungus
In the molecular studies, DNA extraction was carried out using 10–12-day-old culture with Qiagen Blood and Tissue Kit. PCR protocol was undertaken using ITS1 and ITS4 gene region. The positive isolates were sent to the sequencing facilities. The sequence results were opened software program called BIOEDIT, and nucleotide sequences were blasted in the NCBI website. As a consequence of blasted sequences that were showed (100%) homology with the other sequences; B. bassiana. The deposited accession numbers on the NCBI website of the 2 B. bassiana isolates are MW295632 and MW295633, respectively.
Bioactivity of entomopathogenic fungi
First, bean leaves were cut and placed on moist filter papers in a 3-cm Petri dish; then, 1 ml suspension of each of BGF14 and BCA32 with 1×105, 1×106, 1×107, and 1×108 conidial concentrations was sprayed on the leaves, using a spray tower device (Burkard Manufacturing Co. Ltd., Rickmansworth, Herts, UK) (Kumral et al. 2010). Petroleum jelly was applied to edges of the leaves that were left to air dry for 30 min. Afterwards, T. urticae females (1–2 days old females were tested) were placed on the leaves using a paint brush for each dose. The control group was sprayed with water containing 0.02% Tween 80 on leaves, using a spray tower device. The experiments were carried out at room temperature of 24 ± 1 °C, RH of 65 ± 1%, and 14:10 photoperiod. The experiments were monitored after 3, 5, and 7 days post-application, and the ratios of dead and alive mites were recorded using a stereomicroscope. Each dose had 4 replicates (60 tested individuals), and the experiments were repeated 3 times for BGF14 and twice for BCA32.
Data analysis
The data obtained in the study was converted to measurements’ percentage, transformed using arc-sin transformation, and then analyzed with analysis of variance (ANOVA). Percentage mortality was corrected by Abbott (1925). Comparisons between the percentage mortality were made using Duncan’s multiple comparison test. The LC50 and LT50 values were calculated according to Finney (1971) in POLO-PC package program. All statistical analyses were done in SPSS 23.0 package program (IBM Corp, 2013).