Howard T. Dulmage

CORRELATION BETWEEN SPECIFIC PLASMIDS AND DELTA-ENDOTOXIN PRODUCTION IN BACILLUS THURINGIENSIS

Abstract Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry−) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized ( Gonzalez and Carlton, 1980 ). The plasmid patterns of both Cry− and Cry+ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry− mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry− mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (~130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.

A proposed standardized bioassay for formulations of Bacillus thuringiensis based on the international unit

Abstract Since standardization of preparations of Bacillus thuringiensis containing the δ-endotoxin cannot be accomplished by spore count alone, a bioassay is needed. However, before a bioassay can enjoy widespread use, there must be a generally accepted bioassay procedure. A bioassay based on the international unit (IU) and using the cabbage looper, Trichoplusia ni , is proposed to fulfill this requirement. In this assay, the product is incorporated into an alfalfa meal-based diet, and 15–25 mg larvae are allowed to feed on the diet for 5 days. At the end of the test period, activity is measured by determining the LD 50 of the test material and comparing it with that of a standard preparation. Potency is then expressed as IU/mg.

Insecticidal activity of HD-1, a new isolate of Bacillus thuringiensis var. alesti.

Abstract Preparations from fermentation beers of HD-1, a new isolate of Bacillus thuringiensis var. alesti , were 200 times more active in laboratory tests against the pink bollworm, Pectinophora gossypiella , the tobacco budworm, Heliothis virescens , and the cabbage looper, Trichoplusia ni , than a typical commercial formulation, and were 20 times more active than an improved commercial product now undergoing tests. The activity of two preparations of HD-1 and the commercial products in the three test species was also compared against the proposed international standard, E-61, produced by the Institut Pasteur, Paris, France. One lot of HD-1, coded HD-1-PL-1, was adopted as a secondary standard; it had a potency of 16,000 IU/mg. The homology of the international standard to the four test materials was considered.

Coprecipitation with lactose as a means of recovering the spore-crystal complex of Bacillus thuringiensis

Abstract Although the spore-crystal complex of Bacillus thuringiensis can be precipitated from concentrated aqueous suspensions derived from fermentation beers by the addition of acetone, the spores and crystals in the resulting products have a strong tendency to clump, and they are difficult to resuspend in water. Clumping can be reduced by suspending the concentrated complex in 4–6% lactose solutions and precipitating the lactose along with the complex by the addition of 4 vol of acetone. The precipitate is then easily recovered as a dry, stable preparation that is not difficult to resuspend in water. Yields of over 70% were obtained, and the ratios of spores to crystals did not change during the process. A new term, the diet dilution unit, was defined and used to express potencies and calculate yields.

Bioprocess Developments in the Production of Bioinsecticides by Bacillus Thuringiensis

Perspectives technologiques de la lutte contre les insectes. Aspects economiques. Applications sur le terrain. Caracteristiques generales. Parametres de fermentation. Besoins concernant la formulation. Procedes commerciaux de production

Production of the spore-δ-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media

Abstract The spore-δ-endotoxin complex of Bacillus thuringiensis was recovered from beers produced by 12 variants of this bacterium grown in two fermentation media. Activity could not be predicted from the variant, the amount of growth of the organism, or the medium. Also, the total amount of δ-endotoxin recovered and its concentration in the final product differed from variant to variant and between the two media. Activity, measured by bioassays, did not correspond with spore counts. Therefore, studies on the production of the δ-endotoxin in the B. thuringiensis fermentation will require the use of a bioassay. Similarly, the potency of the formulation of the B. thuringiensis -δ-endotoxin can be determined only by bioassay and cannot be predicted from serotype, fermentation medium, or spore count.

Production of δ-endotoxin by eighteen isolates of Bacillus thuringiensis, serotype 3, in 3 fermentation media

Abstract The spore-δ-endotoxin complex of Bacillus thuringiensis was recovered from beers produced by 16 isolates of B. thuringiensis var. alesti (serotype 3a) and by 2 isolates of B. thuringiensis var. kurstaki (serotype 3a, 3b). The amount of endotoxin produced by the isolates varied over a wide range and depended on the isolate used and the medium on which it was grown. Thus, the activity of preparations of B. thuriengiensis could not be predicted from the serotype. Five isolates produced higher yields of the δ-endotoxin than any previously reported.

Interactions between the tobacco budworm, Heliothis virescens, and the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki: Relationship between length of exposure to the toxin and survival

Abstract Larvae of the tobacco budworm, Heliothis virescens, which were fed ad libitum for 24, 48, and 72 hr on a diet treated with various levels of the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki and then transferred to an untreated diet, showed an unexpected capacity to recover from the effects of the toxin, although, as the length of exposure increased, the capacity decreased. Observations on larvae held to emergence indicated that recovery from the toxin was complete. X-ray studies using Ba2+ incorporated into the diet showed that, although the toxin paralyzed the midgut of the treated animals, many animals recovered after the toxin was removed, with food once again passing through the gut.

Novel fermentation media for production of δ-endotoxins from Bacillus thuringiensis☆

Abstract Novel approaches were pursued in the development of practical media for δ-endotoxin production by Bacillus thuringiensis subspp. kurstaki and entomocidus . Several agro-industrial by-products, including cottonseed meal, fish meal, beef blood, slaughterhouse residues, corn steep liquor, and sorter liquor, were investigated for their abilities to support toxin production by these varieties. In addition, fodder yeasts and a variety of low-priced plant proteins available in Egypt, exemplified by such leguminous seeds as horse beans, kidney beans, lima beans, soybeans, chick peas, lentils, and peanuts were incorporated in fermentation media as sole sources of proteins for biosynthesis of the endotoxins. The cotton pests, Spodoptera littoralis, Spodoptera exigua , and Heliothis armigera , were used as test insects for biological assays of spore-δ-endotoxin formulations derived from the novel fermentation media. Fodder yeast, beef blood, and slaughterhouse residues were among the by-products yielding good sporulation titers and potent spore-δ-endotoxin preparations. Formulations of subsp. kurstaki produced from media containing these nutrients killed 80–100% of larvae of H. armigera when tested at 500 μg/ml diet. Most of the formulations derived from fermentations using leguminous seeds as sole sources of protein also contained high levels of spores and endotoxin. For example, LC 50 values determined against S. littoralis of spore-endotoxin preparations of subsp. entomocidus grown in media containing kidney beans, chick peas, or peanuts were 93.4, 93.4, and 110.0 μg/ml diet, respectively. The application of these findings to the practical use of B. thuringiensis is discussed.

Occurrence of two serologically distinct groups within Bacillus thuringiensis serotype 3 ab var. kurstaki.

Abstract Two groups distinguishable on the basis of crystal serology have been identified within Bacillus thuringiensis var kurstaki (serotype 3 ab). The toxicities of these two groups to Trichoplusia ni and Heliothis virescens expressed as T H ratio also differed. It is suggested that serotype 3 ab be separated into two subgroups designated as K-1 and K-73.