Sampling
Ninety soil samples were collected from a depth of about 20 cm. Before collection, the surface layer of soil was removed. A total of five soil samples was taken from each collection site and placed in a plastic bag and transported to the laboratory. The five collected soil samples of each site were placed together in a plastic bag and mixed completely. Small stones and some rough parts were removed (Sevim et al. 2010a). Finally, 1-g soil sample was used to isolate EPFs.
Isolation of Beauveria and Metarhizium spp.
One gram of a given soil sample and 10 ml of the sterilized distilled water were mixed in 15-ml test tubes, which were vortexed for 10 min to obtain homogenous solution. Then, a serial dilution from 10−1 to 10−7 for each soil sample was prepared to isolate a single colony of fungi. Two hundred fifty microliters of the obtained soil extracts from each tube was spread on selective medium (SDA (Sabouraud Dextrose Agar) containing 0.2 μg/ml dodine (N-dodecylguanidine monoacetate), 100 μg/ml chloramphenicol, and 50 μg/ml streptomycin sulphate) and incubated at 28 °C for 2 weeks (Goettel and Inglis 1997). At the end of the incubation period, growing single colonies were transferred on other SDA plates to get pure cultures. One hundred microliters of conidial suspension (1 × 105 conidia/ml) for each fungal isolate was plated on PDAY (1% yeast extract and potato dextrose agar) and incubated at 28 °C for 1 week to select colonies derived from a single colony. The selected colonies from a single colony were transferred on another PDAY for further investigation. All purified fungal isolates were stored in 15% glycerol at − 20 °C.
Identification of fungal isolates
Firstly, fungal isolates were morphologically characterized based on some fungal structures obtained from monosporic pure cultures. Colony morphologies, conidia, and conidiogenous cell shapes were used for initial characterization. All isolates were identified based on the identification key of Humber (1997). Morphological identification of the isolated fungi was confirmed by molecular characterization, using gene sequences of the nuclear intergenic region (bloc) and beta-tubulin (Bt). Genomic DNAs were extracted from monosporic fungal cultures, using the microbial DNA isolation kit (MO-BIO, Carlsbad, CA, USA) according to the manufacturer’s recommendations. For Beauveria isolates, approximately 1500-bp segments of bloc gene region was amplified by the primer pairs of B5.1F (5′-CGACCCGGCCAACTACTTTGA-3′) and B3.1R (5′-GTCTTCCAGTACCACTACGCC-3′) as described in the paper of Rehner et al. (2006). PCR conditions were adapted according to Rehner et al. (2006). For Metarhizium isolates, an approximately 1300-bp segments of Bt gene region were amplified by PCR, using the primer pairs of T1 (5′-AACATGCGTGAGATTGTAAGT-3′) and T22 (5′-TCTGGATGTTGTTGGGAATCC-3′) based on O’Donnell and Cigelnik (1997). PCR amplifications for Bt gene region were performed in a total volume of 50 μl, which included 5 μl Taq DNA polymerase reaction buffer, 3 μl MgCl2, 1.5 μl 10 pmol of each primers, 1.5 μl 10 mM dNTP mix, 1 μl 5 U/μl of Taq DNA polymerase, 1 μl genomic DNA, and 35.5 μl d H2O. Reactions were first incubated for 5 min at 95 °C, and then, 35 cycles were performed as follows: 1 min at 94 °C, 1 min at the annealing temperature of 55 °C, and 2 min at 72 °C. Reactions were then incubated at 72 °C for another 5 min.
After performing all amplifications, PCR products were separated on 1.0% agarose gel and stained with ethidium bromide. The amplified PCR products were viewed under UV light to see correct amplification. Finally, PCR products were sent to the MACROGEN for sequencing. The sequencing process was performed by amplification primers. The obtained sequences were used to carry out phylogenetic analysis.
For Beauveria isolates, the isolated fungi were compared to the known Beauveria species, using bloc sequences according to Rehner et al. (2011) to confirm correct identification. For Metarhizium isolates, the isolated fungi were compared to the fungal isolates and species used in the study of Bischoff et al. (2009) using Bt sequences.
Bioassay experiment
For bioassay experiment, eight EPFs were used in laboratory bioassay experiment to determine their pathogenicity against C. pomonella larvae. All isolates were initially plated on PDAY, using the conidial concentration of 1 × 106 conidia/ml to obtain the monosporic fungal cultures and then incubated at 28 °C for 1 week. A single colony derived from a single spore for each isolate was transferred to another PDAY agar and incubated at 28 °C for 3 weeks. Conidial suspensions were obtained by adding 10 ml of 0.01% Tween 80 as a wetting agent to each Petri dish and gently scraping the surface of the cultures with a sterile cell scraper. After that, conidial suspensions were filtered through two layers of sterile cheesecloth into a 15-ml test plastic test tube to remove rough parts of medium and mycelia. Conidia were counted using an improved Neubauer hemocytometer. Aqueous suspensions applied in bioassays were prepared by dilution at concentration of 1 × 108 conidia/ml. Conidial viability was determined by spreading 100 μl of a 1 × 106 conidia/ml suspension onto the surface of PDAY. After incubation at 28 °C for 24 h, the numbers of viable and non-viable conidia were counted under a microscope. Conidia were considered to have germinated if the germ tube was longer than the diameter of the conidium. Isolates with a viability of above 95% were used for bioassay experiments (Sevim et al. 2010b).
A conidial concentration of 1 × 108 conidia/ml was used to determine the pathogenicity of all tested isolates against C. pomonella larvae. For the bioassay experiment, the healthy C. pomonella larvae were obtained from laboratory culture at Ahi Evran University, Genetic Bioengineering, and Microbiology Laboratory. Healthy larvae were randomly selected and used for the bioassay. For each isolate, 10 larvae were directly immersed in the conidial suspension for 5 s. Another 10 larvae were immersed in 0.01% Tween 80 solutions as a control group. The treated and control larvae were separately placed into plastic boxes (20 mm) containing freshly prepared artificial diet with ventilated lids to permit airflow. Ten larvae were used for each replicate, and all experiments were repeated three times on different occasions. Finally, all boxes were incubated for a period of 2 weeks at 25 °C in darkness. The test larvae were checked daily to record mortality rate. At the end of the incubation period, all dead larvae were surface sterilized with 2% sodium hypochlorite solution for 2 min, followed by 70% ethanol for 2 min, and washed two times in sterile distilled water for 30 s. After that, they were placed into the moisture chamber to stimulate fungal sporulation outside the cadaver to confirm infection by the tested fungi.
Data analysis
The obtained sequences of complimentary strands were edited and aligned with Clustal W contained within Bioedit version 7.1.3.0 (Hall 1990; Thompson et al. 1994). The partial sequences of bloc gene regions from Beauveria isolates used in this study and reference isolates from the study of Rehner et al. (2011) were combined, using Bioedit. For Metarhizium isolates, Bt gene sequences from this study and the study of Bischoff et al. (2009) were combined and used for phylogenetic analysis. Phylogenetic analyses, using the maximum likelihood (ML) method, were performed by PHYML software packed in SeaView version 4 by selecting the general time-reversible (GTR)-based substitution matrix and gamma distribution (Guindon et al. 2010; Gout et al. 2010). The reliability of the dendrogram was tested by bootstrap analysis with 1000 pseudoreplicates, using SeaView version 4. Mortality data were corrected according to Abbott’s formula (Abbott 1925). Differences between the fungal isolates and the control group, with respect to mortality and mycosis, were determined by analysis of variance (ANOVA) and subsequently by Dunnett’s one-tailed t test. Differences among the fungal isolates with respect to mortality rates and mycosis were determined by ANOVA and subsequently by LSD multiple comparison test. All analyses were performed by using SPSS 16.0 statistical software.
GenBank accession numbers
The DNA sequences obtained from this study were deposited in NCBI GenBank database. For Beauveria isolates (ELA-1, ELA-2, ELA-4, ELA-5, ELA-6, ELA-7, ELA-8, ELA-9, ELA-11, ELA-13, ELA-14, ELA-15, ELA-16, ELA-18, ELA-24, ELA-25, ELA-26, ELA-27, ELA-28, ELA-29, ELA-30, ELA-31, ELA-32, ELA-35, ELA-36, ELA-37, ELA-40, ELA-41 and ELA-42), accession numbers are from MH181833 to MH181861. For Metarhizium isolates (ELA-3, ELA-10, ELA-12, ELA-17, ELA-19, ELA-20, ELA-21, ELA-22, ELA-23, ELA-33, ELA-34, ELA-38 and ELA-39), accession numbers are from MH181862 to MH181874.
