ISOLATION, CHARACTERIZATION, AND CULTURE OFBACILLUS THURINGIENSISFROM SOIL AND DUST FROM GRAIN STORAGE BINS AND THEIR TOXICITY FORMAMESTRA CONFIGURATA(LEPIDOPTERA: NOCTUIDAE)

Abstract

AbstractOne hundred and twoBacillus thuringiensisBerliner strains isolated from six different types of Canadian soil and dust from different grain storage bins were cultured in shake flasks containing Great Northern White Bean (GNWB) protein concentrate (48.6% protein) as the main nitrogen source and dextrose as the main carbohydrate source. The resulting endotoxins were bioassayed against the bertha armyworm,Mamestra configurataWlk. Thirty-three percent of soil and 66% of grain dust samples were positive forB. thuringiensis. The bacterium was found most frequently in organic-rich soil. The four most toxic soil isolates (which were seven to 15 times more toxic than the international standard, HD1-S-1980), and two nontoxic grain dust isolates were characterized by serological typing, biochemical analysis, carbohydrate utilization, plasmid profile analysis, protein profile analysis using sodium dodecyl sulfate – polyacrylamide gels, and polymerase chain reaction. Four isolates were determined to be subsp.kurstakicontaining 130–140 and 63–65 kDa proteins, and two isolates (tested for comparison) were subsp.canadensiscontaining 31 and 38 kDa proteins. Nonpyramidal-crystal-producing strains did not grow well in culture media containing GNWB, degossypellized cotton seed meal (61% protein), defatted soy flour (55% protein), or peptone as nitrogen sources. Excess of GNWB protein concentrate in shake flask culture media (30 g/L) inhibited bacterial growth and reduced the toxicity of isolate A1.2/72 subsp.kurstaki, which was the most toxic soil isolate. Isolate A1.2/72, which was 15 times more toxic for bertha armyworm larvae than the international standard (HD1-S-1980), contained threecry1Agenes (cry1Aa,cry1Ab, and cry1Ac), whereas HD-1 lacked thecry1Abgene. This strain was synergistic with strain HD-551 subsp.kenyae(cry1A,cry2A, andcry1Bgenes) but not with HD-133 subsp.aizawai(cry1Ab,cry1B,cry1C, andcry1Dgenes) when the strains were cultured together in a cotton seed meal medium and fed toM. configurata. The growth rate, economic yield, and toxicity of the new isolate, A1.2/72, produced in a 14-L laboratory fermenter declined when the fermentation ingredients were tripled. We believe that the indigenous strain A1.2/72 warrants further research development for bertha armyworm control.