Screening And Partial Characterisation Of δ-Endotoxins From Some Local Bacillus Thuringiensis Isolates For Insecticidal Activity Against The Spotted Stem Borer.

ABSTRACT

Prolonged use of synthetic chemical pesticides is environmentally undesirable, results in rapid development of resistance among insect pests and is economically unmanageable by small-scale farmers. Kenya has unexplored potential in controlling lepidopteran pests 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, present in some Kenyan Bt isolates, characteristic of Cry1 and/or Cry2 proteins, their efficacy as affected by different temperatures and their specificity on an invasive and prevalent lepidopteran stem borer, Chilo partellus (Swinhoe). Delta–endotoxin crystals containing insecticidal Cry proteins were isolated and purified from cultures of 20 unidentified local Bt isolates using froth floatation and centrifugation. Total protein in the resulting suspensions was quantified using Bradford assay method and protein yield estimated at 3.14 ±0.084 mg/ml of nutrient broth culture with a purity level of 54.8 % ±15.3 %. The efficacy of the δ-endotoxins against C. partellus was evaluated using leaf–dip bioassays. Among the isolates evaluated, Bt 44 and Bt 48 had the most potent δ-endotoxin crystals towards the 1st instar larvae, causing mortality of 62.6 % and 64.8 % respectively after 72 h. The effect of the δ-endotoxins’ concentration and temperature on larval mortality was assessed for 72 hours at temperatures of 24°C, 27°C and 31°C and concentrations of 0.01 mg/ml, 0.1 mg/ml and 1.0 mg/ml. The resulting LC50 were 52.3 µg/ml and 42.0 µg/ml whilst LT50 values were 76.7h and 60.9h for Bt 44 and Bt 48 respectively. Higher efficacy was observed at 24°C and 31°C than at 27°C, an indication that these δ-endotoxins are suited for local conditions where higher temperatures are experienced than in temperate regions. Interaction between concentration and temperature was significant for δ-endotoxins of Bt 48 but not those of Bt 44. Electrophoresis using SDSPAGE revealed a major protein component of the δ-endotoxins had a molecular weight Mr ~ 130 kDa which was digested to a trypsin-resistant core of Mr ~ 70 kDa. Cry protein analysis using ELISA detected more Cry1 in Bt 44 than Bt 48 δ-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. Chromatographic analysis using RP-HPLC revealed some differences in the elution profiles of δ-endotoxins of both Bt 44 and Bt 48, an indication that there may be different types and quantities of the Cry toxins in the crystals or even novel proteins. These results indicate that the two local Bt isolates expressed Cry1 and probably Cry2 proteins that are capable of controlling C. partellus and may therefore be utilised as sources for δ-endotoxins for biopesticide development for controlling the pest.