Collection of soil samples
A total of seventy (70) soil samples were collected from Dalota, Kality, Erobgebaya, Koka, and Menagesha forest sites, Ethiopia. Samples were collected by scraping off the surface soil, and from each site, approximately 1.5 kg of soil samples were collected from 2 to 5-cm depth and placed in alcohol-sterilized polyethylene plastic bags. Collected soil samples were brought to the Applied Microbiology laboratory and stored at 4 °C for further processing.
Isolation of entomopathogenic bacterium Bt from soil
A modified version of the temperature selection method was used to isolate Bt from soil samples as described by Travers et al. (1987). Soil samples (10 g) were individually mixed in 100 ml sterile water and homogenized with an orbital shaker (200 rpm) for 4 h at room temperature. After complete homogenization, the samples were prepared to appropriate dilution from which samples were taken and heated at 80 °C for 10 min in a water bath equipped with a shaker to destroy non-spore formers and vegetative Bacillus cells. Then, 0.1 ml soil of aliquot samples was individually inoculated into nutrient agar and incubated at 28 °C for 48–72 h. Colonies were randomly picked and subcultured on nutrient agar and maintained for further investigation.
Cultural characterization
A total of 280 bacillus colonies were picked and characterized for their cultural characteristics such as colony’s color, shape, margin, elevation, and surface. Bacterial colonies showing typical Bt were preliminary identified based on cultural and morphological characteristics regarding Bergey’s Manual of Determinative Bacteriology (Halt et al. 1994).
Morphological characterization
Gram staining
Gram staining of isolates was carried out following the protocol of Provine and Gardner (1974). Thin bacterial smear was made on a clean glass slide, dried in the air, and heat-fixed. The smear was covered with crystal violet and kept for 1 min. The slide was washed, covered with Gram’s iodine, and let stand for 1 min. The slide was washed, decolorized with acetone, rocking the slide gently. The decolorization step was done very quickly. Immediately, the slide was washed with water, counterstained with safranine and let stand for 30 s, washed with water, blot dried, and examined under the microscope (× 100 magnifications).
Coomassie brilliant blue staining
The presence of parasporal bodies of isolates was detected, using Coomassie brilliant blue (CBB) staining, following the protocol of Rampersad et al. (2002). Selected isolates were inoculated into a sterile 50-ml conical flask containing nutrient broth and incubated in an orbital shaker (250 rpm) for 90 to 110 h at room temperature. Samples were smeared onto glass slides, followed by an air dry and heat fixing. Then, the slides were stained using 0.133% Coomassie blue stain in 50% acetic acid for 5 min. The dried slides were then observed under a phase-contrast microscope (× 100) to visualize the formation of parasporal bodies and spores.
Preparation of bacterial suspension
Isolates from nutrient agar slants were transferred into the fresh nutrient broth and incubated at 28 °C aerobically for complete sporulation and lysis of all vegetative cells. Completion of sporulation and lysis (spore-crystal formation) was detected by using a hemocytometer with the aid of a phase-contrast optical microscope (Soares-da-Silva et al. 2015). The spore-crystal mixture of each isolate was standardized at a concentration of 1 × 109 spores/ml for bio-assay.
Preliminary pathogenicity screening of bacterial isolates using G. mellonella under laboratory condition
Rearing of Galleria mellonella
Rearing of larvae of Galleria mellonella L. (Lepidoptera, Pyralidae) was undertaken according to Meyling (2007) at the Ambo Plant Protection Agricultural Research Center. Adult moths were kept in 500-ml flasks containing folded tissue paper to facilitate their mating and egg-laying potential. Eggs were laid on folded tissue paper, and each tissue paper was transferred from a flask into rearing plastic containers containing honey, wheat bran, and glycerol as dietary components for hatching larvae. The plastic containers were incubated at 20 °C for 4 weeks under darkness. The resulting third to fourth instar larvae were used for bio-assay evaluation.
Bio-assay evaluation
Insecticidal activities of isolates were preliminarily bio-assayed on the G. mellonella larvae, using the diet contamination technique (Navon et al. 1990). From each culture, 5 ml of spore-crystal suspension containing 1 × 109 spores/ml prepared and mixed with 15 g artificial diet containing honey, wheat bran, and glycerol. Then, 10 larvae of G. mellonella were transferred into an artificial diet containing a spore-crystal suspension mixture for feeding. The same number of larvae in an artificial diet without spore-crystal suspension was included as a control. All experiments were repeated 4 times. Periodic mortality was recorded every 48 h for 144 h.
Molecular characterization of entomopathogenic bacterial isolates
DNA extraction
The genomic DNA of isolates was extracted by the heat shock method using TE buffer (Ricieto et al. 2013). Isolates were grown on the nutrient agar at 30 °C for 15 h. Collected 10–20 mg bacterial isolates from the growing colony of approximately 1–2 mm in diameter was transferred to 200 μl of Tris–EDTA buffer (10 mM Tris; 1 mM EDTA; pH 8.0), using a sterile toothpick. The suspension was homogenized and incubated for 10 min in a boiling water bath (100 °C) for lysis of bacterial cells. Then, the suspension was centrifuged at 10,000g for 5 min to remove debris. The supernatant was transferred to a new microcentrifuge tube and stored at − 20 °C for PCR amplification reactions (polymerase chain reaction), and 5 μl of the supernatant was used as a template in the PCR reaction.
PCR amplification
The amplification of 533 bp groEL gene of Bt isolates was carried out, using B. cereus group-specific primer BalF (5′-TGCAACTGTATTAGCACAAGCT-3′) and BalR (5′TACCACGAAGTTTGTTCACTACT-3′) as described by Chang et al. (2003). Five microliters of the template was amplified in 25 μl of reaction mixture consisting of 10 mM Tris-HCl (pH-9), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 1 U Taq DNA polymerase (Fermentas), and 10 pmol of respective primers. The PCRs were performed using a thermocycler (Mastercycler, Eppendorf, Germany) using BalF and BalR primers consist of 30 cycles of 94 °C for 45 s (denaturation), 55 °C for 45 s (annealing), and 72 °C for 45 s (extension). The amplified PCR products were analyzed in 1.5% agarose gel prepared in TAE buffer (40 mM Tris-acetate and 1 mM EDTA, pH 8.0) containing ethidium bromide (0.3 μg/ml). Electrophoresis was carried out at 7 V/cm for 1 h and 30 min in a submarine gel electrophoresis system (Bangalore Genei, India), and the gel was photographed by UV gel documentation system (Alpha Innotech Corporation, USA). The PCR product was purified by using the QIAquick PCR purification kit according to the manufacturer’s instructions (QIAGEN, Germany) and sequenced. Finally, sequenced data were submitted to NCBI (National Center for Biotechnological Information) and compared to publish sequence of NCBI database for the identification of bacterial isolates at the species level.
In vitro bioassay evaluation of bacterial isolates against B. tabaci
Rearing of whiteflies
Whitefly adults were collected from Koka in the tomato field using a manual aspirator. The area is characterized by a mean minimum and maximum temperature of 12.14 °C and 27.39 °C, respectively Ethiopian sugar development agency (ESDA 2010). After collection, insects were reared on young tomatoes planted in pots in whitefly-proof cages (Safavi and Bakhshaei 2017). Tomato seedlings with 2 or 4 leaves were placed into a cage and allowed to be infested by adult whiteflies (sex ratio ≈ 1:1). Adults were allowed to oviposit for 24 h on leaves. Then, adults were removed, and plants with whitefly eggs were transferred to other insect-free cages. Whitefly eggs were allowed to develop into the nymphal stage, and 3rd and 4th nymphal instars were applied in bioassays
Single-concentration bioassay
Virulent isolates were evaluated against the nymph of B. tabaci under laboratory conditions with the leaf disc method (Ateyyat et al. 2009). The leaf discs with 3rd to 4th nymphal instars were cut from plants and immersed in 1 × 109 spores/ml concentration for 10 s and placed upside down onto 0.2% plain water agar medium in Petri plates. Four replicates (Petri dishes) were set per treatment. Nymphs were considered dead if body color changed from yellowish to dark brown or if the body appeared dry. Periodic mortality was recorded every 48 h for 144 h. Isolates with mortality greater than 50% were selected to estimate the median lethal concentration (LC50).
Multiple-concentration bioassays
The multiple-concentration bioassays were evaluated to estimate LC50, following the leaf disc method as before (Ateyyat et al. 2009). For this study, four (4) highly virulent isolates were selected and evaluated. Leaf discs with 3rd to 4th nymphal instars were immersed in the 5 concentration ranges from 1 × 105 to 1 × 109 spores/ml of each isolate. For each concentration, 4 repetitions were performed, and periodic mortality was recorded every 48 h for 144 h.
Effect of temperature on the growth of isolates
The growth rate response of isolates to different temperatures was evaluated as described by Hamedo (2016) with a little modification. Isolates were inoculated on nutrient broth and incubated for 24 h. One milliliter of each broth culture (cell suspensions) was inoculated into 50 ml sterilized nutrient broth in a 250-ml Erlenmeyer flask and incubated at 15, 20, 25, 30, 35, and 40 °C. The vegetative growth phase of isolates was determined by the growth curve experiment. The maximum growth rate of isolates was measured by taking optical density reading at 600 nm in 6-h intervals starting from 0 h, using a spectrophotometer. Optical density reading and colony-forming units (CFU) were used to plot the regression line, and the maximal growth rate of each isolates in response to different temperatures was calculated.
Data analysis
Mortality data were corrected using Abbot’s formula (Abbott, 1925), arcsine transformed, and subjected to the ANOVA procedure of SPSS version 20.0. Means were separated using Tukey’s honestly significant difference (HSD) at 5% significance level for screening experiments against G. mellonella and B. tabaci, respectively. The LC50 and LC90 values of isolate were determined by using probit analysis (Finney, 1971).