Mechanisms for Selective Toxicity of Locally Isolated strains of Bacillus Thuringiensis (Berliner).

Abstract

Bacillus thuringiensis is a gram-positive soil-dwelling bacterium. It produces a crystalline insecticidal protein (delta-endotoxin), during the sporulation phase of its growth. Several strains have been isolated and the specificity in its insecticidal action on different hosts is an attractive aspect for its use in biological control. Investigations were carried out into the fermentation of two isolates, M37 /2 and TIKKI. In order to optimize the culture conditions in shake-flask cultures, a range of media were studied with respect to bacterial growth and o-endotoxin production (i.e. biological activity). Of all the media tested, M3 and M4 recorded highest production of M37/2 and TIKKI respectively. Biological activity did not correlate to total bacterial numbers.Physical studies were carried out on three isolates of B. thuringiensis: TIKKI,MF48/2 and M37 /2. Each has insecticidal activity against Glossina species (Oiptera: Glossinidae), Chilo partellus Swinhoe (lepidoptera: Pyralidae) and Spodoptera exempta Walker (lepidoptera: Noctuidae), as well as Culicine and Anopheline mosquitoes. Electrophoretic separation of crystals from the three isolates revealed major bands of Mr - 140-25 kd. Further detailed study on the TIKKI isolate showed that the crystal had two main b~nds of Mr- 120 kd and 66 kd . Solubilized crystal (protoxin) revealed a band of Mr - 64 kd while the activated toxin was approximately Mr- 62 kd . Immunological experiments using antibodies raised against TIKKI toxin gave a strong cross reactivity -viiibetween TIKKI and MF48/2 isolates, but not with M37 /2 isolate. Brush border membrane vesicles (BBMV) and soluble proteins prepared from miguts of G. m.centralis Machado and C. partellus were used to study the interaction with 8.thuringiensis protoxin/toxin. The Protoxins/toxins were 1251-radioiodinated.Studies with BBMV from C. partellus showed three bands of Mr- 68 kd, 64 kd and 28 kd for MF4B/2 isolate. A single band of Mr- 64 kd was observed for G. m. centralis and TIKKI isolate. Homologous competition experiments showed that labelled toxin retained its ability to interact with BBMV even in the presence of a 100-fold higher concentration of unlabelled toxin. Gel filtration techniques using solubilized midgut proteins incubated with 1251-toxin showed interaction with detergent extracted aqueous protein but not with buffer soluble proteins. Histopathological studies showed that all cells (except the giant cells of the mycetome) were susceptible to both TIKKI and MF4B/2 toxins, with the latter being more pathogenic. M37/2 toxin was not toxic to midgut cells. The extent of cell damage can be related to length of exposure to, as well as, the concentration of the toxin . Studies carried out on the inhibito"iy-effects of 8.thuringiensis crystal from TIKKI isolate on the activity of midgut membrane ATPase showed a non-competitive inhibition. The V max of the enzyme activity was effectively lowered by > 50%. The concentration of K + ion in the A TPase assay medium was found to affect activity with a peak at 80 Mm. The concentration of Na+ ion on the other hand had no apparent effect on ATPase activity.