Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisors: Clifford Keil and Vincent D'Amico, Dept. of Entomology & Wildlife Ecology. Includes bibliographical references.

To develop an assay to detect antibodies to Bacillus thuringiensis subsp. israelensis in mallard ducks, a growth curve was first established for the bacterium. The growth curve indicated that the crystal delta endotoxin would be best harvested from the rest of the cell material after 12 hours of growth. The delta endotoxin was solubilized in alkaline conditions followed by treatment with proteases or no treatment. The two differently treated delta endotoxins were purified by column chromatography. Fractions were assayed for duck erythrocyte lysis and cytotoxicity to a mosquito cell line. The proteolyzed sample gave four protein peaks with gel filtration, and the fourth peak containing biological activity was further separated into three protein fractions by anion exchange chromatography; two of the three showed biological activity. These two fractions contained 22 and 23 kD proteins species. The nonproteolyzed sample was separated into two protein fractions by gel filtration; only the first peak contained the biological activity. This fraction was further separated into two fractions by anion exchange chromatography; only the second fraction, containing a 28 kD protein, exhibited the activity. This fraction contained a 28 kD protein. However, the fractions containing 22 or 23 kD proteins originating from the proteolyzed sample showed the highest biological activity. Mallard ducks were repeatedly exposed to an aerosolized commercial preparation of the organism. Sera were collected periodically and tested for the antibody by an enzyme-linked immunosorbent assay (ELISA). Those toxic antigens containing 22 or 23 kD proteins were unsuitable for the assay. The exposed ducks were found to produce antibodies against the first fraction from anion exchange chromatography of the proteolyzed sample. The antibody titres increased as the number of exposures increased. The results suggest that ELISA is applicable for detecting antibodies against B.t.i. in wild ducks using the fraction containing a 50 kD protein. / Graduation date: 1992

La ecología de Bacillus thuringiensis, un bioinsecticida muy común, es poco conocida. Nuestro principal objetivo era investigar acerca de la ecología de esta bacteria en la filosfera. En un primer estudio se recogieron 35 muestras de hojas del género Piper de bosques andinos colombianos. Se obtuvieron 256 aislamientos de B. thuringiensis del 74% de las muestras estudiadas. Los aislamientos fueron caracterizados según la morfología del cristal, la presencia de genes cry por PCR y la toxicidad contra insectos. Además, se estimaron las poblaciones de células vegetativas viables y endosporas por unidad de área obteniéndose 2-5x103 cfu/cm2 de hoja. En general, no se encontraron diferencias estadísticamente significativas en el número de aislamientos de B. thuringiensis por cm2 de hoja ni en las características de los aislamientos de B. thuringiensis entre las diferentes especies de Piper. Después de comprobar que B. thuringiensis estaba presente en el filoplano, se quiso comparar las poblaciones de esta bacteria en el suelo y en las hojas. Se aisló B. thuringiensis del filoplano y del suelo de cultivos de maíz y fríjol. Se recuperaron 214 aislamientos de 96 muestras de filoplano (0-34 cfu/cm2) y 59 aislamientos de 24 muestras de suelo. Todos los aislamientos fueron caracterizados como se ha explicado anteriormente. Las poblaciones predominantes de B. thuringiensis en el filoplano contenían genes cry1 y eran activas contra Spodoptera frugiperda, mientras que los aislamientos del suelo tenían genes cry11 y eran activos contra Culex quinquefasciatus. El hecho de que predominaran poblaciones específicas de B. thuringiensis en las hojas diferentes a las del suelo sugiere que existe una selección diferencial en las poblaciones de B. thuringiensis en el filoplano y en el suelo. Entonces, se investigó la capacidad de migración de B. thuringiensis desde el suelo a las hojas. Se inocularon dos cepas diferentes de B. thuringiensis en suelos, semillas y hojas jóvenes de plantas de fríjol, para determinar si podían migrar a las hojas superiores en condiciones controladas. Aunque se recuperaron aislamientos de B. thuringiensis en las hojas, las poblaciones fueron muy bajas (menos de 10 cfu/cm2 de hoja). Además el número de células recuperado disminuía a medida que las hojas estaban más distantes del suelo o de las hojas inoculadas. Todo esto indicaba que B. thuringiensis migra pobremente desde el suelo o la semilla a las hojas o entre hojas de la misma planta. También se evaluó la capacidad de varias cepas de B. thuringiensis de colonizar la superficie de las plantas y se comparó con otras bacterias epífitas. Mientras que todas las cepas de B. thuringiensis se multiplicaron en cierta medida después de la inoculación sobre hojas de fríjol, las poblaciones máximas alcanzadas fueron de 106 cfu/g de hoja, muy inferiores a las conseguidas por otras bacteria epífitas, como Pseudomonas fluorescens. Muy poco tiempo después de la inoculación, una porción importante de las células de B. thuringiensis estaban en forma de endospora. Además el crecimiento de B. thuringiensis no se vio afectado por la presencia de Pseudomonas spp. cuando fueron co-inoculados y viceversa. Por otro lado, cuando se observaron al microscopio las cepas de B. thuringiensis (marcadas con el gen de la proteína verde fluorescente) sobre hojas de fríjol, se observó que no formaban agregados celulares y no estaban asociadas con otras bacterias epífitas ni con estructuras de la hoja. Finalmente se investigó la capacidad de B. thuringiensis de crecer en un medio diseñado para simular la composición de nutrientes del filoplano. Sin embargo el crecimiento fue inferior al de otras bacterias. Aparentemente, B. thuringiensis tiene unos requisitos nutricionales mayores que otras especies bacterianas habitantes naturales del filoplano. / The ecology of Bacillus thuringiensis, a common biopesticide, is poorly understood. Our main objective was to investigate the ecology of this bacteria on the phylloplane. In a first study 35 leaf samples of the genus Piper were collected from the Colombian Andean forest. Two hundred and fifty-six isolates of B. thuringiensis were obtained from 74% of the samples studied. The isolates were characterized by crystal morphology, the presence of cry genes by PCR and toxicity against insects. The populations of viable vegetative cells and spores per unit area were estimated (2-5x103 cfu/cm2 of leaf). Overall, no significant differences in the number of B. thuringiensis isolates per cm2 of leaf nor in the B. thuringiensis characteristics were found among the different Piper species evaluated. After observing B. thuringiensis on the phylloplane, a comparison was performed between soil and leaf populations. B. thuringiensis was isolated from the phylloplane and soil in plantings of maize and bean from Colombia; and 214 isolates were recovered from 96 phylloplane samples (0-34 cfu/cm2) while 59 isolates from 24 soil samples. All the isolates were characterized as above-mentioned. The predominant population of B. thuringiensis on the phylloplane harbored cry1 gene and was active against Spodoptera frugiperda, whereas in soil the isolates harboring cry11 gene and active against Culex quinquefasciatus predominated. The predominance of specific B. thuringiensis populations both on the leaves and in the soil, suggests the presence of differential selection in B. thuringiensis populations on the phylloplane and in soil. Then, we addressed the process of immigration of B. thuringiensis from soil to leaves. Two different B. thuringiensis strains were inoculated into soils, onto seeds or onto lower leaves of bean plants to determine if they were able to disperse to upper leaves under controlled conditions. While B. thuringiensis isolates were commonly recovered from leaves exposed to such inocula, populations were very low (less than 10 cfu/cm2 of leaf). In addition, the number of cells of B. thuringiensis recovered decreased with increasing distance from the soil or from the inoculated leaves. This indicates that B. thuringiensis disperses poorly from the soil or the seed to the leaves or between leaves of the same plant under controlled conditions. Moreover, the ability of several B. thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 106 cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain. Many cells were in a spore form soon after inoculation onto plants. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae spp. when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures when directly observed on bean leaves by epifluorescent microscopy. Finally, we analyzed the capacity of B. thuringiensis to grow on a medium designed to simulate the nutrient composition of the phylloplane but the growth observed was very poor compared to other bacteria. This bacterium apparently has greater nutrient requirements than other bacterial species that are prominent inhabitants of the phylloplane.

Resistance to Bacillus thuringiensis (Bt) cotton, Gossypium hirsutum, is associated with fitness costs in pink bollworm, Pectinophora gossypiella. Previous work has demonstrated that costs of resistance are induced by environmental factors including insecticidal cotton phytochemicals such as gossypol. This study (1) tested the hypothesis that the presence of toxic phytochemicals would increase the dominance and magnitude of fitness costs associated with genes conferring resistance to the Bacillus thuringiensis (Bt) toxin, (2) coupled a selection experiment and simulation modeling to evaluate the fitness cost of resistance to Bt in pink bollworm and (3) used laboratory strains containing susceptible and resistant genotypes to test the hypothesis that gossypol accumulates more readily in pink bollworm with Bt resistance alleles, and that this gossypol accumulation reduces fitness. To test hypothesis (1), larval survival and weight were measured in two independent strains of pink bollworm, Pectinophora gossypiella, reared on diet containing the cotton phytochemicals gossypol and cyclopropenoid fatty acid, alone, or in combination. Cotton phytochemicals increased the dominance and magnitude of fitness costs associated with cadherin-based resistance to Bacillus thuringiensis (Bt) toxin. Gossypol and cyclopropenoid fatty acid combined had a more detrimental effect on fitness of pink bollworm than either compound alone. To test hypothesis (2), two pink bollworm strains fed synthetic diet were monitored over 30 generations to test the hypothesis that costs associated with Bt resistance would result in a decline in the frequency of resistance. A decrease in resistance allele frequency did occur in both strains and costs affecting each resistant genotypes were estimated. To test hypothesis (3), two strains of pink bollworm were fed on diet containing gossypol and on diet without gossypol, and gossypol accumulation in tissues of genotypes was measured. In both strains, significantly more gossypol accumulated in genotypes containing at least one resistance allele and gossypol accumulation was additive to dominant. In both strains, an increase in the dominance or magnitude of costs affecting larval weight was observed on gossypol diet, and the change in the magnitude of costs was positively associated with gossypol absorption. In one strain, the presence of gossypol increased survival costs but only in the genotype with the highest gossypol absorption. The mutation conferring resistance to the Bt toxin Cry1Ac is found in the cadherin-encoding region in three lepidopeteran pests (Helicoverpa armigera, Heliothis virescens and Pectinophora gossypiella) including the pink bollworm. Cadherin proteins had been hypothesized to play a role in maintaining the integrity of the insect midgut epithelial tissue. Taken together, these results support the hypothesis that cadherin proteins do contribute to gut integrity, toxic plant phytochemicals accumulate more readily in pink bollworm with resistance alleles, and that such accumulation increases the dominance and magnitude of fitness costs.

Various strains of Bacillus thuringiensis Mt.) produce crystalline endotoxins specific for larvae of different insect classes. Two strains, B.t. subspp. israelensis and kurstaki produce similar 130 kDa toxins encoded by the CryIVB gene (toxic to Diptera) and the CryIA gene (toxic to Lepidoptera), respectively. The N-terminal region of the CryIVB gene was cloned into the Escherichia coli expression vector pKX223-3. A mosquitocidal transformant was obtained as determined by mosquito bioassays. The gene fragment, if stable, can be cloned into cyanobacteria to achieve biological control of mosquito-borne diseases. A second goal was to identify the binding specificity domain of the CryIVB gene which encodes the portion of the protein toxin that binds the insect midgut causing cell lysis and death. Two potential insect binding specificity domains identified by computer analyses were switched with a known binding specificity region of the CryIA gene. The polymerase chain reaction was utilized to obtain gene fragments of the CryIVB gene which replaced the CryIA gene binding specificity domain. The resulting recombinant clones carrying the CryIA gene containing the .000nd proposed insect binding specificity domain of the CryIVB gene were fotsd to be mosquitocidal. / Department of Biology

Thesis (Ph. D.)--University of Adelaide, Dept. of Crop Protection, 1998? / Includes bibliographical references (leaves 120-145).

Dissertation (Ph.D.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

Techn. Univ., Diss.--Braunschweig, 2000.

Universiẗat, Diss., 1980 u.d.T.: Eppler, Birgit: Aggregation von Bacillus cereus und Chlorella vulgaris in Gegenwart von Natriumionen und Kalziumionen--Karlsruhe.

Techn. Hochsch., Diss., 2004--Aachen.