Sample collection and isolation of EPFs
From July to October 2019, infected O. japonica adult cadavers were collected from three kiwifruit orchards planted in Kemalpaşa District of Artvin Province, Turkey. First, the surface of the specimens was disinfected in 1% sodium hypochlorite solution for 1 min, rinsed with sterile water, and blotted dry. The dried specimens were placed in 9-cm-diameter Petri dishes containing potato dextrose agar (PDA; BD Difco, Sparks, MD, USA) medium supplemented with streptomycin sulfate (0.5 g l–1) and incubated at 24 ± 1 °C for 7–10 days in the dark. Before identification studies, the single spore isolation of isolates was then performed using the serial dilution method. (Dhingra and Sinclair 1995). The EPF isolates were transferred to pieces (1 cm2) of Whatman no. 1 filter papers overlaid on PDA. After colonization, the filter papers were dried and stored in the Eppendorf tubes at − 20 °C for further use and long-term storage (Erper et al. 2016). The strains of B. bassiana were deposited in the culture collection of the Mycology Laboratory of the Plant Protection Department, Faculty of Agriculture, Ondokuz Mayis University (Accession nos: KA-78-1-14).
Isolation of genomic DNA and molecular identification
Molecular tools, including phylogenetic analysis based on the internal transcribed spacer (ITS) and 26S large subunit (LSU), accurately identify EPF isolates. Conidial masses were gathered from the surface of 7-day-old PDA cultures and ground into powder in liquid nitrogen. According to the manufacturer’s instructions, genomic DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). The quality and quantity of obtained DNA were measured spectrophotometrically by the A260/A280 ratio using a DS-11 FX + nano spectrophotometer (Denovix Inc., Wilmington, DE, USA). The DNA extract was diluted to 10 ng/μl and stored at − 20 °C for further analyses.
The ITS and LSU regions of ribosomal DNA were amplified with primer sets ITS1/ITS4 (White et al. 1990) and LR5/LR0R (Vilgalys and Hester 1990). The PCR mixture contained 1 × PCR reaction buffer, 1.25-unit of Ampliqon TEMPase Hot Start DNA polymerase (Berntsen, Rodovre, Denmark), 0.2 mM of each dNTP, 0.4 µM of each primer, 10 ng of template DNA, and molecular grade water up to 50 μl. The PCR amplification was conducted at 95 °C for 15 min for an initial cycle to denature DNA and activate Taq DNA polymerase, followed by 95 °C for 45 s, annealing at 54 °C for 60 s, and extension at 72 °C for 90 s for 35 cycles; and a final extension at 72 °C for 10 min. The amplicons were bidirectionally sequenced by Macrogen Inc., sequencing service (Seoul, Republic of Korea).
The DNA sequences were edited, and consensus sequences were manually estimated using Mega X computing platforms (Kumar et al. 2018). All sequences were compared against the GenBank nucleotide database (https://www.ncbi.nlm.nih.gov/genbank/) using the BLASTn algorithm, and deposited in GenBank. The isolates from this study, with additional reference isolates (Fig. 1) retrieved from the GenBank database, were aligned in the MAFFT v.7 online interfaces (Katoh and Standley 2013), http://mafft.cbrc.jp/alignment/server/) using default settings. A maximum likelihood (ML) tree of the combined ITS and LSU data set was inferred using the command-line version of IQ-TREE 1.6.7 (Nguyen et al. 2015) run on the CIPRES Science Gateway V 3.3. (Miller et al. 2010, https://www.phylo.org/), with ultrafast bootstrapping implemented with 1000 replicates. The resulting trees were analyzed and edited in FigTree v1.4.2 software. The ITS and LSU sequences of Metarhizium anisopliae strain CBS 662.67 were added as an-outgroup to facilitate the generation of consensus trees.
Determination of mycelial growth and conidiation
Mycelial growth and sporulation of 14 isolates were evaluated according to Cheng et al. (2016). Mycelial discs (5-mm-dia.) from 7 to 10-day-old fungal cultures were placed in the center of Petri dishes (9-cm-dia.) containing PDA, and the dishes were sealed by a parafilm and incubated at 25 ± 1 °C for two weeks. Their mycelial growth was measured on days 4, 8, 12, and 16 at two perpendicular radii of the colony, and their first day of sporulation was recorded. To determine the sporulation per unit area, at the end of the 16 days, an agar piece of 1 cm2 for each isolate was cut from cultures where fungal growth occurs with a sterile scalpel and placed into 50-ml sterile polypropylene tubes. The conidia were released from the agar piece by shaking and dispersed in 20 ml of sterile distilled water containing Tween 20 (0.02%) (polysorbate, Merck Millipore KGaA, Burlington, USA). The conidia were then counted under a light microscope (DM1000, Leica Microsystems, Wetzlar, Germany) at 400× magnification using a hemocytometer, and the spore concentration per unit area was calculated. Four replicates were performed for each isolate, and the experiment was conducted once.
Adults of O. japonica were collected directly with a mouth aspirator from plants of kiwifruit (Actinidia deliciosa) cv. Hayward in 10-yr-old commercial orchards in the Artvin Province, on June. Insects were placed into a plastic box (30 × 40 × 25 cm) with a perforated lid for aeration, transported to the laboratory, and maintained in a climate chamber at 25 ± 1 °C for two days with a 16:8 h L:D photoperiod. Insect cultures were fed with fresh Acacia leaves (Acacia sp.), and after two days, healthy adults were gently selected for bioassays.
Inoculum of entomopathogen fungal isolates
Fourteen B. bassiana isolates were cultivated on PDA at 25 ± 1 °C for 15 days in darkness. Conidia were harvested with 100 ml of sterile distilled water containing Tween 20 (0.02%) and homogenized with a magnetic stirrer for 10 min. Then, mycelia were removed by filtering conidia suspensions through four layers of sterile cheesecloth. Conidial suspensions were counted under the DM1000 light microscope using a hemocytometer to calibrate five concentrations of each isolate (1 × 104, 105, 106, 107, and 108 conidia ml–1) (Erper et al. 2016). To determine the conidial germination rate (%), 100 μl conidial suspension (1 × 104 conidia ml−1) of each isolate was spread on PDA in 6-cm-diameter Petri dishes and incubated at 25 ± 1 °C. After 24 h, germinated conidia were counted by examining 100 conidia from four different areas in each dish, using the DM1000 light microscope at 400× magnification. Conidia were regarded as germinated when they produced a germ tube, at least half of the conidial length. The experiment was three replicated for each isolate.
Concentration–time response bioassay
The concentration (1 × 108 conidia ml–1) of all B. bassiana isolates prepared in sterile distilled water containing Tween 20 (0.02%) in advance was used to determine the concentration–time relationship. The bottoms of 500 ml plastic cups (10.5 × 8.5 × 6.0 cm), which 1.0% NaOCl disinfected, were covered by filter paper moistured with sterile-distilled water. Then, 10 adults of O. japonica were placed in the cups having fresh Acacia leaves. The concentration of each isolate was applied to adults (2 ml per cup) using a hand sprayer. Only sterile-distilled water containing Tween 20 (0.02%) was sprayed to control plastic cups, and the cups were incubated at either 20 ± 1 °C or 25 ± 1 °C at 75 ± 5% RH and 16:8 h L:D photoperiod. Inspections were made daily until individuals died in all the cups. Dead individuals were counted under a stereomicroscope (EZ4, Leica Microsystems, Wetzlar, Germany) at 40× magnification. The mortality rate was recorded daily, and dead individuals were removed from the cups. To determine the mycosis rate, evidence of Beauveria on which the fungal sporulation was observed on adult cadavers was verified by microscopic inspection after seven days (Boston et al. 2020). Three replicates of ten adults (n = 30) were used for each isolate, and the experimental design was completely randomized. Finally, the mean mortality rate was corrected for control mortality by Abbott’s formula (Abbott 1925), and the percentage of mycosed cadavers was calculated.
Concentration–mortality response bioassay
Concentration-mortality bioassay trials were assayed using four isolates of B. bassiana based on their high efficacy on adults of O. japonica in the concentration–time bioassays. Five concentrations from 104 to 108 conidia ml–1 of the isolates were evaluated to determine the concentration–mortality relationship and the median lethal concentration (LC50). O. japonica adults were exposed to these five concentrations of each isolate at 25 °C and sterile distilled water for control as the concentration–time response bioassay. The number of dead adults was documented for five consecutive days after applying conidial concentrations. The bioassays were conducted once, with three replications (n = 30). Finally, Abbott’s formula was used to correct the mean mortality rate for control mortality, and the median LC50 values were calculated using probit analysis.
Since mortality rates exceeded 5% in pathogenicity tests, Abbott’s formula was used to correct these data. Independent-time mortality data expressed (50% lethal time LT50) and (LT90) values from bioassays were calculated by fitting the data by using Logprobit method with Probit analysis program POLO-PLUS ver. 2.0 (Robertson et al. 1980). The LT50 and LT90 values of the isolates were compared using confidence intervals (95%), and the slopes of the regression lines were compared using standard errors. Data obtained from the present study were separately analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s Honestly Significant difference (HSD) at P < 0.05 using SPSS software version 14.0.1 for Windows (SPSS Inc., Chicago, IL, USA).
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