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The effect of aeration, initial pH,l-cystine concentration, supplemental sodium chloride, and Tween 60 (polyoxyethylene sorbitan monostearate) in culture media on biomass production and potency ofBacillus thuringiensissubsp.aizawai(HD133) was studied in shake-flask cultures. HD133 had a high oxygen requirement and a starting pH of 7.0 for optimal biomass production and spore–crystal toxicity. HD133 had a low tolerance for the amino acid,l-cystine. The addition of 0.5% NaCl and 0.1% Tween 60 to a culture medium containing cotton seed meal (Proflo) and glucose as the main nitrogen and carbohydrate sources, respectively, enhanced the potency of the spore–crystal product. This medium was judged superior to five others in laboratory tests for biomass production and potency of HD133.

We suggest a new mechanism for the maintenance of specificity of the association between the entomopathogenic nematode Steinernema scapterisci and its symbiotic bacteria. We evaluated the development and reproduction of infective and non-infective juvenile S. scapterisci in monoxenic combinations with its symbiotic bacteria, Xenorhabdus sp. ‘S’ and with the bacterial symbiont of Steinernema carpocapsae and Steinernema riobravis. Although development of non-infective stages occurred on all Xenorhabdus spp., the development of infective juveniles to the 4th stage (‘dauer’ recovery) was significantly delayed and reduced with X. nematophilus and Xenorhabdus sp. ‘R’, the bacterial symbionts of S. carpocapsae and S. riobravis, respectively. ‘Dauer’ recovery improved significantly when the cultures of X. nematophilus and Xenorhabdus sp. ‘R’ were supplemented with cell-free filtrates from Xenorhabdus sp. ‘S’. The infective juvenile S. scapterisci produced in all 3 cultures were virulent to Galleria mellonella larvae, confirming successful retention of Xenorhabdus from other steinernematids in their intestine. In fact, S. scapterisci infective juveniles containing X. nematophilus or Xenorhabdus sp. ‘R’ were more virulent to G. mellonella than those containing their natural symbiont, Xenorhabdus sp. ‘S’. We believe that this is the first demonstration of the symbiont-specific exit of infective juveniles from the ‘dauer’ phase which represents the finest level of specificity of bacteria–nematode association. This is also the first report of successful isolation of the natural symbiont of S. scapterisci.

AbstractOne hundred and two Bacillus 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 for B. 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 three cry1Agenes (cry1Aa, cry1Ab, and cry1Ac), whereas HD-1 lacked the cry1Abgene. This strain was synergistic with strain HD-551 subsp. kenyae(cry1A, cry2A, and cry1Bgenes) but not with HD-133 subsp. aizawai(cry1Ab, cry1B, cry1C, and cry1Dgenes) when the strains were cultured together in a cotton seed meal medium and fed to M. 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.