Insect culture
A laboratory culture of P. xylostella was established initially from pupae obtained from National Bureau of Agricultural Insect Resources, Bangalore, India. The pupae were transferred to the rearing cages (45 × 45 × 45 cm) for the emergence of adults. The newly emerged adults of P. xylostella were fed on 10% sugar solution, and mustard seedlings were provided as oviposition substrate and the hatched larvae were fed by fresh cauliflower leaves (CFL-1522, Syngenta) maintained under greenhouse conditions without any insecticide application. The larvae were placed in a (30 × 30 × 30 cm) larval rearing cage with a glass top, wooden bottom and mesh on all 4 sides. The insect culture was maintained at the Insect Bioassay laboratory (Department of Plant Biotechnology, TNAU, Coimbatore, India) under controlled conditions of temperature, humidity and photoperiod (25 ± 1 °C, 75 ± 5% RH and 16: 8 h (L: D).
Bt isolates and growth conditions
Sixty native Bt isolates were obtained from Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, TNAU, Coimbatore, India. The reference strain Bacillus thuringiensis subsp. Kurstaki HD1 originally obtained from Bacillus Genetic Stock Center (Columbus, Ohio) was used. In order to obtain single colonies, all the isolates were subcultured on T3 agar media plates using the quadrant streak method and incubated at 30 °C for 12–14 h. Single colony was picked using sterile loop and transferred to the test tube containing 5 ml of T3 broth, incubated at 30 °C for 24 h at 200 rpm and stored as sterile 50% glycerol stocks at − 20 °C for further use.
Isolation of spore–crystal mixture for toxicity analysis
The spore crystal mixture was isolated from 58 Bt isolates and a reference strain, HD1 as a positive control and an acrystalliferous strain 78/11 as a negative control. To obtain the spore crystal mixture from each isolate, a single colony of Bt culture from T3 agar plates was inoculated into culture tubes containing 5 ml of T3 broth and kept for overnight incubation in a shaking incubator (Orbitek, Scigenics Biotech Pvt. Ltd, Chennai, India) maintained at 30 °C and 200 rpm. From the overnight grown cultures, 1% inoculum (250 µl) was added to the 250-ml conical flask containing 25 ml of T3 broth in a shaking incubator maintained at 30 °C, 200 rpm for 48–60 h. The growth and lysis of the bacterial cells were checked using the phase contrast microscope after 48 h. When more than 90% cells have lysed, culture was centrifuged at 4 °C for 10 min at 10,000 rpm (Centrifuge 5810R, Eppendorf, Germany) and resulting pellet was suspended in 25 ml of ice-cold Tris–EDTA buffer [Tris 10 mM, EDTA 1 mM, pH 8.0 with 1 mM phenyl methyl sulphonyl fluoride (PMSF)] and washed once with 25 ml of ice-cold 0.5 M NaCl and centrifuged for 10 min, followed by 2 washes with 25 ml Tris–EDTA buffer with 0.5 mM PMSF at the same speed and time (Ramalakshmi and Udayasuriyan 2010). Finally, the pellet was suspended in 500 µl of sterile distilled water containing 10 µl of 1 mM PMSF and stored at − 20 °C as aliquots of 50 µl for later use.
Colony and crystal morphology
Colony morphology pertaining to colour, surface and margin of each single bacterial colony was observed from the culture plate maintained at 30 °C for 24 h. To examine the shape of the parasporal crystalline inclusions using glass slides, culture smears were prepared on a glass slide, heat fixed and stained with Coomassie brilliant blue stain (0.133% Coomassie Brilliant Blue G250 in 50% acetic acid) for about one min. Smears were washed gently in running water, blot-dried and observed through the phase contrast microscope.
Protein analysis
Protein analysis using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was done for spore crystal mixture from all the Bt samples. SDS-PAGE was performed by following the standard protocol (Laemmli 1970), using 10% separating gel and 4% stacking gel. The spore crystal mixtures were mixed by loading buffer (4x) containing 0.25 M Tris HCl pH 6.8, 8% SDS, 40% glycerol, 0.5% bromophenol blue in the ratio 4:1, respectively. Then, samples were boiled for 2 min before loading into wells. The molecular mass of the proteins was estimated using pre-stained three-colour protein ladder (Puregene, Genetix Biotech Asia Pvt. Ltd.) covering a wide range molecular weight from 10 to 315 kDa.
Detection of cry genes in Bt isolates
Total genomic DNA was isolated from a single bacterial colony of all isolates according to method described by Sambrook and Russell (2001) and used as a template for the amplification of cry1, cry2, cry9 and vip3A1 genes. The isolated DNA was quantified through NanoDrop (Genova Nano, Jenway), and the integrity was evaluated by agarose gel electrophoresis (1%). Each polymerase chain reaction (PCR) mixture contained 20–50 ng template DNA, 1 µM of each primer (Ben-Dov et al. 1997) and 10 µl of 2 × PCR Master Mix (Smart prime) consisting of dNTPs, Taq polymerase and PCR buffer, and the final volume was made up to 20 µl with sterile distilled water. PCR amplification was performed in a thermal cycler (ProFlex PCR system, Applied Biosystems). The PCR amplicons were separated in 1% agarose gel using 1 kb DNA ladder. The amplified products were visualized under UV transilluminator (Bio-Rad).
Insect toxicity assays with Bt isolates
Insecticidal activity of the spore crystal mixtures of all the Bt isolates was tested against 3-day-old P. xylostella larvae. In order to conduct the bioassay, the protein concentration was estimated using Bradford method (Bradford 1976) and the suspension was diluted to 25 µg/ml of concentration in all the isolates before conducting toxicity tests. In all the cases, spore crystal mixture was smeared on small discs (25 mm diameter) of cauliflower leaves and allowed to dry. To prevent desiccation, all the leaf discs were placed on the wet surface of Whatman filter paper discs in plastic cups (30 mm diameter). For each isolate, 3 replications with 10 larvae per replicate were used. The standard strain, Bt subsp. kurstaki (Btk) HD1 and acrystalliferous strain 78/11 were used as positive and negative controls, respectively. Insect mortality was recorded at 24-h interval for 3 days, and cumulative mortality at 72 h was taken as mortality for comparing isolates.
Statistical analysis
Experiments were carried out in a completely randomized design (CRD). Single-dose bioassays were statistically analysed by one-way analysis of variance (ANOVA) using AGRES statistical software version 7.01, and significant differences between means were determined by Duncan’s multiple range test (p = 0.05).
