Indigenous Bt isolates and growth conditions
A total of 50 indigenous Bt isolates were obtained from Bt laboratory stock, Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India (Additional file 1: Table S1). The reference strains for a different group of Cry toxins originally received from Bacillus Genetic Stock Center (BGSC, Columbus, Ohio) were used as a positive check. The Bt cultures were revived from glycerol stocks on T3 agar medium (per litre: 3.0 g Tryptone, 2.0 g Tryptose, 1.5 g Yeast extract powder, 6.9 g NaH2PO4; 8.9 g Na2HPO4 and 0.005 g MnCl; 10.0 g agar (pH 6.8–7.0) and incubated at 30 °C for 14 h. In order to get single colonies, the overnight grew Bt cultures were subcultured on T3 agar medium by quadrant streak method and incubated at 30 °C for overnight. Purified single colonies were inoculated separately in test tubes containing 5 ml of T3 broth and incubated at 30 °C for 24 h with 200 rpm (Orbitek, Scigenics Biotech India Pvt Ltd., Chennai, India).
Bacterial colony and crystal morphology
The colony morphology of individual Bt isolates was examined visually. A loop full of Bt culture was inoculated in 5 ml of T3 broth and incubated at 30 °C for 48 h at 200 rpm. To check the spore-crystal inclusions in the Bt isolates, a loop full of culture smears was prepared in glass slides, heat-fixed, and stained with the 0.133% Coomassie Brilliant Blue stain (G250). Then, the stained-glass slides were washed gently with water, blot dried with tissue paper, and observed under a bright-field microscope for the existence of crystalline inclusion (Leica DM 1000LED, DFC295, Germany).
Preparation of spore-crystal mixtures from Bt isolates
A single colony of Bt cultures was inoculated into 5 ml of T3 broth and incubated at 30 °C overnight at 200 rpm. After the overnight incubation, 1% grown cultures were inoculated into a 250-ml conical flask containing 25 ml of T3 broth and incubated at 30 °C with shaking of 200 rpm for 48–60 h. The bacterial sporulation was observed under a bright-field microscope (Leica DM 1000LED, DFC295, Germany). When more than 90% of cells were lysed, the sporulated cultures were transferred into 50-ml falcon tubes and incubated at 4 °C for half-an-hour before harvest. The sporulated bacterial cultures were centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 25 ml of ice-cold Tris–EDTA buffer (10 mM Tris; 1 mM EDTA and pH 8.0) containing 1 mM PMSF (phenyl methyl sulphonyl fluoride) and washed thrice with Tris–EDTA buffer and once with 25 ml of ice-cold 0.5 M NaCl solution containing 0.5 mM PMSF by centrifugation at 8000 rpm for 10 min at 4 °C (Eppendorf centrifuge 5810R, Germany). The final pellet was dissolved in nuclease-free water containing 1 mM PMSF and stored at − 20 °C (Ramalakshmi and Udayasuriyan, 2010).
Protein profiling of Bt isolates
Protein profiling was done using SDS-PAGE (Sodium dodecyl sulphate poly acrylamide gel electrophoresis) by the method of Laemmli (1970) using 10% separating and 4% stacking gel. The spore crystal mixture samples were prepared by mixing with 4× loading dye (0.25 M Tris HCl pH 6.8; 8% SDS, 40% glycerol, 0.5% bromophenol blue) in a ratio of 4:1. Then, the samples were incubated in boiling water for 2 min before loading. The molecular weight of the protein was estimated by using three colour pre-stained protein marker (PG PMT 2922, Puregene, Genetix Biotech Asia Pvt Ltd.,) covering a broad range of molecular weights from 10 to 250 kDa.
Bacterial genomic DNA isolation and polymerase chain reaction
Purified single colonies of Bt isolates were used for genomic DNA isolation by following Sambrook and Russell’s (2001) method. Extracted genomic DNA was used as a template DNA (30–50 ng) for the amplification of cry1, cry2, cry3, cry4, cry9, vip1, vip2, and vip3 genes with their specific primers (Additional file 1: Table S2). A total reaction volume of 20 µl consists of 1 µl of template DNA, 1 µM of each primer, 10 µl of 2X PCR master mix (SmartPrime) consisting of dNTPs, Taq polymerase, and 7 µl of nuclease-free water. PCR analysis was performed in Mastercycler Nexus GX2 (Eppendorf, Germany), and the PCR products were resolved in agarose gel electrophoresis with ethidium bromide staining. The products were visualized under a UV trans-illuminator for the expected size of amplification of different cry and vip genes (Additional file 1: Table S2).
Insect cultures
The insect cultures of Plutella xylostella (pupae), Spodoptera litura (eggs), S. frugiperda (eggs), and Helicoverpa armigera (eggs) were obtained from NBAIR (National Bureau of Agricultural Insect Resources, Bangalore, India) and established in insect bioassay laboratory at Department of Plant Biotechnology, CPMB&B, TNAU, Coimbatore, India. The newly emerged adult P. xylostella was supplemented with a 10% sugar solution along with Vitamin E, and two leaf-stage mustard seedlings were provided as an oviposition substrate. Newly hatched neonate larvae were transferred into Cauliflower leaf (maintained under controlled conditions) and were maintained at the laboratory with fresh cauliflower leaves as and when required. The cultures of S. litura, S. frugiperda and H. armigera larvae were reared on an artificial diet. Pupae were collected and allowed for adults’ emergence in an insect-rearing cage (2.5 × 2.5 Ft). Newly emerged adult moths were supplemented with 10% sugar solution along with Vitamin E. Young maize seedlings for S. frugiperda and H. armigera, and Nerium oleander shoots for S. litura were placed inside the cage as an oviposition substrate. Eggs were collected and maintained in Petri plates for hatching. Newly hatched neonates were transferred into an artificial diet by using a camel hairbrush and maintained.
Toxicity of Bt isolates against lepidopteran insects
The concentration of spore-crystal mixtures was estimated by Broadford’s reagent method (Bradford 1976). Estimated spore-crystal mixtures were equalized to a concentration of 20 µg/ml and used for in vitro insect bioassay. Detached leaf bit bioassay was carried out with insect-specific host plants. Cauliflower leaves were used for P. xylostella and S. litura, while maize leaves were used for S. frugiperda and H. armigera. Circular leaf bits of cauliflower (2 cm dia) were prepared and coated with the spore-crystal mixture on both sides (10 µl/side) of the leaf disc and air-dried. Similarly, 2 cm leaf bits of maize leaves were treated with spore-crystal mixtures (10 µl/side). Treated leaf bits were placed into a 3 cm dia plastic container containing moist filter paper and pre-starved neonate larvae were released. Ten larvae were used per treatment and replicated thrice. The larval mortality was observed up to 72 h after treatment and expressed in percentage. The entire in vitro insect bioassay experiments were conducted under a controlled environment with 25 ± 1 °C and 65 ± 2% RH.
Statistical analysis
The laboratory experiments were performed in a completely randomized design (CRD). The larval mortality data were computed using Abbott’s formula (Abbott 1925), and subjected to one-way variance (ANOVA) in AGRES statistical software version 7.01, and significant differences between means were determined by Duncan’s multiple range test (DMRT) at p = 0.05.
