Toxicity Effects Of Δ-endotoxins Obtained From Native Bacillus Thuringiensis Isolates Against Maize Stalk Borer (Busseola Fusca Fuller)

Abstract

Decrease in maize crop yield due to infestation by the maize stem borer (Busseola fusca) presents a major food security threat in Kenya and many sub-Saharan countries. Farmers in Kenya have relied on the use of insecticides to control B. fusca. However, this approach presents several challenges including negative effects on human health, killing of non-target organisms, widespread pest resistance, and harm to the environment. Bacillus thuringiensis (Bt) produces δ–endotoxins during the sporulation phase, and these proteins have specific toxicity against a wide range of pests including lepidopteran insects. This study was carried out to evaluate the efficacy of δ-endotoxins from local Bt isolates in control of B. fusca, determine the effective dosage, and characterize the proteins from the most effective isolates. The pest neonates were fed on maize leaf discs contaminated with broth of various Bt isolates. A total of 43 isolates, including the standard Bacillus thuringiensis aizawai, were included in the study. The first study involved mortality tests to determine the most effective isolates and a bioassay was used to determine the LD50. Proteins from the most effective isolates were harvested using the centrifugation method and used in a bioassay in which five concentrations of the proteins were applied to the maize leaf discs and fed to the neonates. The effective isolates were characterized to determine their differences using the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and reverse phase high performance chromatography (RP-HPLC) methods. The gel electrophoresis involved running the prepared samples through a gel of matrix concentration of 0.25% w/v and recording the results on a UV transilluminator. RP-HPLC enabled the elucidation of the protein profile and involved running the centrifuged proteins on a HPLC system whose solid phase comprised of Jupiter C18 column and mobile phase comprising of methanol and water at ratio of 70:30 (v/v%). The flow rate was maintained at 1ml/min, 10 MPa and the separation monitored using UV detector. There were temporal differences in the mortalities recorded at different

sampling regimes. The average mortalities increased with increase in time, with the lowest mortality being observed after 24 h (6.6%) and the highest after 120 h (86.4%). Bacillus thuringiensis isolates 25, 30 and 52 showed the highest overall mean mortalities at 73%, 69.5% and 67.5% and 90%, 90% and 87.5%, respectively when sampling was done after 72 h. The 24-hr, 48-hr, 72-hr, 96-hr, and 120-hr sampling regimes were used in this study. The bioassay conducted for 12-hr, 24-hr, 36-hr, and 48-hr sampling times showed that toxicity increased with increase in concentrations. The LC50 for the most effective isolates at 36-hour sampling were 0.003201 mg/ml, 0.00367 mg/ml, 0.000942 mg/ml, and 0.002395 mg/ml for isolates Bta, Bt 25, Bt 30, and Bt 52, respectively. The LD50 values of Bta and Bt 25 were not significantly different (p ≥ 0.05). At these concentrations, more than half of the neonates had succumbed to toxic effects of the toxins. The gel electrophoresis results showed that proteins with a size of 130 KDa existed in the Bt samples together with others of between 70 KDa and 100 KDa. The HPLC results showed that the characteristic peak was attained at 190 nm for all isolates at which the highest peaks had a height of 1.64 × 106 mAU, 6.36 × 106 mAU, 1.66 × 106 mAU, and 1.63 × 106 mAU for Bta, Bt 25, Bt 30, and Bt 52, respectively. Peaks with similar patterns were observed in all the isolates, suggesting that the isolates had similar Cry proteins. The findings of this study suggest that local Bt isolates have great potential for use in managing Busseola fusca and other lepidopteran insects.