Screening and partial characterization of d-endotoxins from some local Bacillus thuringiensis isolates for insecticidal activity against the spotted stem borer

Abstract. Kimani PG, Nyambaka H, Kasina M. 2018. Screening and partial characterization of d-endotoxins from some local Bacillus thuringiensis isolates for insecticidal activity against the spotted stem borer. Biofarmasi J Nat Prod Biochem 17: 21-38. Prolonged use of synthetic chemical pesticides is environmentally undesirable, causing the rapid development of resistance among insect pests. Kenya has unexplored potential in controlling lepidopteran pests by using proteinous delta-endotoxins sourced from local isolates of a naturally occurring bacterium, Bacillus thuringiensis Berliner (Bt). This study attempted to identify the insecticidal proteins in some Kenyan Bt isolates, characteristic of Cry1 and/or Cry2 proteins. It also aimed to test their efficacy as affected by different temperatures and their specificity on an invasive and prevalent lepidopteran stem borer, Chilo partellus (Swinhoe). Delta-endotoxin crystals were isolated and purified from cultures of twenty unidentified local Bt isolates using froth floatation and centrifugation. Total protein in the resulting suspensions was quantified using the Bradford assay method, and the approximate protein yield was 3.14 ±0.084 mg/mL of nutrient broth culture with a purity level of 54.8 % ±15.3 %. Leaf-dip bioassays used for testing the efficacy of the d-endotoxins against C. partellus. Among the isolates evaluated, Bt 44 and Bt 48 had the most potent d-endotoxin crystals towards the 1st instar larvae, leading to mortality of 62.6 % and 64.8 % respectively after 72 h. The effect of the d-endotoxins' concentration and temperature on larval mortality was examined for 72 hours at temperatures of 24°C, 27°C and 31°C and levels of 0.01 mg/mL, 0.1 mg/mL and 1.0 mg/mL. The resulting LC50 was 52.3 µg/mL and 42.0 µg/mL, while LT50 values were 76.7h and 60.9h for Bt 44 and Bt 48, respectively. Higher efficacy found at 24°C and 31°C than at 27°C, an indication that these d-endotoxins are tolerable for local conditions where temperatures are higher than in temperate regions. The relationship between concentration and temperature was significant for d-endotoxins of Bt 48 but not those of Bt 44. A major protein component of the d-endotoxins had a molecular weight Mr ~ 130 kDa, which generates a trypsin-resistant core of Mr ~ 70 kDa. Cry protein analysis detected more Cry1 in Bt 44 than Bt 48 ?-endotoxins and no Cry2 in either. However, cry gene analysis using PCR detected the presence of both cry1 and cry2 genes in the DNA of Bt 44 but none in Bt 51, a negative control from toxicity tests against the pest. The chromatographic analysis revealed some differences in the elution profiles of d-endotoxins of both Bt 44 and Bt 48, an indication that there may be different types and amounts of the Cry toxins in the crystals or even novel proteins. These findings indicate that the two local Bt isolates expressed Cry1, and probably Cry2 proteins can control C. partellus and may, therefore, become promising sources for ?-endotoxins for biopesticide development for controlling the pest.