Two novel strains of Bacillus thuringiensis toxic to coleopterans

Abstract

MATERIALS AND METHODS Bacterial strains. B. thuringiensis H serotype reference strains were obtained from the collection of Dulmage (7) or from the Entomopathogenic Bacteria collection of the Institut Pasteur, Paris, France. Growth. For the production of crystal proteins, B. thuringiensis strains were grown as shake flask cultures in DSMG medium [0.4% (wt/vol) nutrient broth (Difco), 0.5% (wt/vol) glucose, 25 mM K2HPO4, 25 mM KH2PO4, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 10 ,uM MnCl2, 10 ,uM FeSO4] at room temperature (21 to 24°C) for 4 to 5 days until sporulation and cell lysis had occurred. For the preparation of flagella, B. thuringiensis strains were grown as static liquid cultures in nutrient broth (Difco) plus 0.1% (wt/vol) glucose. Colony hybridization. A modified colony hybridization procedure of Grunstein and Hogness (12) was used. Environmental samples, consisting of soil, crop dust, and organic material, were suspended in spore buffer (0.06% [wt/vol] sodium phosphate [pH 7.0], 0.05% [wt/vol] potassium phosphate [pH 6.6], 0.005% [wt/vol] sodium dodecyl sulfate [SDS], 1 ,u1 of 1-octanol per ml; final pH of buffer approximately 7) to a concentration of 1 g of material per 10 ml of buffer, heated for 30 min at 60°C, diluted in spore buffer, and spread on NSM (19) agar medium. After overnight growth at 25°C, colonies were blotted from the agar medium to nitrocellulose filters and treated with 0.5 M NaOH-1.5 M NaCl for 30 min and then with 1 M ammonium acetate-0.02 M NaOH for 30 min. The filters were probed at 65°C in a solution containing 3x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), lOx Denhardt's solution (lx Denhardt's solution is 0.02% [wt/vol] bovine serum albumin, 0.02% [vol/vol] Ficoll, 0.02% [wt/vol] polyvinylpyrrolidone), 200 ,ug of heparin per ml, and 0.1% SDS with the 2.0-kb HindIII-XbaI cryIlIA fragment of strain EG2158 (6) or with 3337

Genetically Engineered Crops: From Idea to Product

“…Initially, a forward genetics approach was used to identify the toxin-encoding gene that conferred the insecticidal properties of a particular Bt strain against the target insect pest. The gene coding for the insecticidal toxin (Bt toxin) was then inserted into a crop plant, making it resistant to feeding damage by the target pest (23,86,99). The advent of advanced sequencing and bioinformatics technologies has changed the nature of Bt toxin discovery, enabling the use of a high-throughput approach to sequence multiple Bt strains in a short time and to analyze the sequence information for predicted Bt toxins (102).…”

A Method of Controlling Corn Rootworm Feeding Using a Bacillus thuringiensis Protein Expressed in Transgenic Maize

“…This hybrid uses a variant Bt Cry3Bb1 insecticidal protein (Donovan et al, 1992). Cry3Bb1 is known to be biologically active against several species within the Coleopteran family Chrysomelidae, including the western corn rootworm, Diabrotica virgifera virgifera LeConte (Rupar et al, 1991). The biological activity of this protein against D. virgifera virgifera suggested its potential use in creating transgenic plants expressing Cry3Bb1 that would confer protection to corn root tissue from larval feeding damage.…”

Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide

“…It is not clear why CryIIIAand CryLA(c)-induced ion channels were more readily observed in black lipid membranes than CryIIIB2or domain I peptide-induced ion channels. Both native toxins CryIIIA and CryIIIB2 have nearly equivalent insecticidal activities against the Colorado potato beetle, L. decemlineata (25), suggesting that ion channel activity alone is not predictive of insecticidal activity. We also consider the possibility that deficiencies in one step of the multistep mode of action of Cry toxins, such as inefficient channel formation, might be compensated for at another step of the mode of action.…”