Bacillus thuringiensis (Bt) is a gram-positive common soil bacterium that produces crystals (Cry) containing proteins that are toxic to certain insects, in particular larvae of Lepidoptera and Diptera. The Bt toxin in the past has been widely used as a bioactive compound for the biological control of mainly lepidopteran pests. Most recently a variety of crops, including cotton and maize, have been genetically modified to express a Bt toxin to confer resistance to lepidopteran pests. However, the effect of abiotic environmental stress, such as drought and heat, which are typical for Africa, on Bt toxin expression in a genetically modified crop has so far not been fully evaluated. This study focuses on the expression and stability of the Cry1Ac insecticidal protein from Bacillus thuringiensis in genetically modified cotton plants under drought and heat stress. These include the physiological and biochemical characterization of the expressed Bt toxin gene under drought stress as well as the biological activity against first-instar larvae of the African cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae). Non-genetically modified cotton (Gossypium hirsutum cv. Opal), as well as genetically modified cotton (cv. Nuopal) expressing the Bt toxin Cry1Ac, were exposed to drought and heat stress. Drought stress was induced by withholding watering plants until the soil moisture content reached 25- 30 % of field capacity. Non-stressed control plants were watered and soil moisture content to 80-100 % of field capacity was maintained. For heat stress, plants were grown at 38 to 32 DC during the day and night, respectively, whereas control plants were grown in a growth cabinet at a 28/25 DC day/night cycle. For growth analysis plants were harvested every second week after planting. At each harvest, different parts of the plant were collected and their fresh and dry weight determined. For biochemical analysis and determining biological activity against first-instar larvae of H. armigera, two types of experiments were carried out, the first experiment four weeks after treatment induction and the second experiment eight weeks after treatment induction. Different plant material (leaves, flowers and immature green bolls) were used for Bt detection as well as for determining biological activity against first-instar larvae of H. armigera. Under drought stress conditions a reduction in leaf area and leaf dry weight were found in both Bt toxin expressing and non-expressing cotton plants, but no significant difference in physiological performance between Bt-expressing and non-expressing cotton plants was found. This study shows that the Bt toxin (Cry1Ac) level decreases in senescent plants and that drought stress did not affect the growth and development of genetically modified Bt plants when compared to non-Bt plants. Although the expression of Bt toxin (Cry1Ac) in Bt cotton plants decreased under drought stress no effect on the efficacy of the toxin against H. armigera was observed. In addition, no significant decrease of Bt toxin content was found in Bt cotton leaves after exposure to heat stress when compared to leaves from nonheat stressed plants. / Dissertation (MSc)--University of Pretoria, 2008. / Plant Science / unrestricted

There are apprehensions that genetic modification of maize with Bacillus thuringiensis (Bt) may have negative effects on soil biodiversity, ecosystem processes and functions. This study aimed at determining the effect of Bt maize crop, Bt maize residues and its genetic modification on microbial biomass carbon (MBC), selected enzyme activities, vesicular arbuscular mycorrhizal (VAM) fungi and N and P release patterns. The study was conducted under field, glasshouse and laboratory conditions. In 2010/2011 season, four maize cultivars; DKC 61-25B (Bt), DKC 61-24 (non-Bt), PAN 6Q-321B (Bt) and PAN6777 (non-Bt) were planted. Determination of MBC, enzyme activities and fungal spore count was done at 42, 70, and 105 days after planting (DAP). A loam soil amended with Bt or non-Bt maize leaf residues from a study of 2009/2010 season was incubated to investigate effects of Bt maize residues on MBC and soil enzyme activities. Leaf residues of Bt and non-Bt maize cultivars (DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777) were used and soil without residues was used as a control. Samples were collected at 7, 28 and 56 days of incubation (DOI). An incubation study was also carried out in the laboratory to determine the effect of Bt maize residues (i.e. leaf, stem and root) and its genetic modification on N and P release patterns. Residues of DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777and soil without residues as a control were incubated in the laboratory. After destructive sampling at 0, 7, 14, 28, and 56 DOI, N in the form of NH4-N and NO3-N and P mineralisation were determined. Amendment of soil with residues enhanced MBC (p < 0.05) at all the sampling dates. For example MBC increased from 95 in the control to 146.3 mg/kg in the DKC 61-25B treatment at the end of the glasshouse trial. In the field DKC 61-25B had 9.1 mg/kg greater MBC than DKC 61-24, while PAN 6Q-321B had 23.9 mg/kg more MBC than PAN6777 at the end of the trial. However, no differences (p < 0.05) were observed in enzyme activities under field and glasshouse conditions except for dehydrogenase that had greater activity where DKC 61-25B and PAN 6777 were grown. There were no differences between the type of residues (Bt and non-Bt) on enzyme activities tested. However, differences were observed among the sampling dates. No effects of Bt maize crop on fungal spore count were observed. Similarly no differences were observed in leaf, stem and root tissues composition between Bt and non-Bt maize cultivars. Net N and P mineralisation from Bt maize cultivars did not differ from that of non-Bt maize cultivars. However, differences were observed among the cultivars. The results of this study suggested that Bt maize with Bt MON810 event can be grown in the central region of the Eastern Cape (EC), South Africa without affecting MBC, soil enzyme activities, VAM, and release of N and P nutrients from its residues.

There are concerns that genetic modification of maize with Bacillus thuringiensis may influence its structural and chemical composition which, together with the Cry1Ab proteins, may affect agro-ecosystem processes and feed quality. This study was aimed at investigating the expression of Cry1Ab protein in leaves, stems and roots of Bt maize and the effect of genetic modification (MON810) on activity of earthworms, silage quality and decomposition of residues in the field. In 2009/10 four maize cultivars; DKC61-25B, DKC61-24, PAN6Q-321B and PAN6777 were planted. Expression of Cry1Ab in leaves, stems and roots was analyzed at 6, 8, 10 and 12 weeks after planting (WAP). Earthworms were also sampled from the same treatments at 6, 9 and 18 WAP. Two silage experiments were conducted using maize cultivars, DKC80-12B and DKC80-10 produced in the 2008/09 season and DKC61-25B, DKC61-24, PAN6Q-321B and PAN6777, produced in 2009/10. The silage was sampled at 0, 2, 4, 8, 15 and 42 days in 2008/09 and 0, 8 and 42 days in 2009/10 and analyzed for Ash Free Dry Matter, Crude Fiber, Neutral Detergent Fiber, Acid Detergent Fiber, Acid Detergent Lignin, Crude Protein and Total Digestible Nutrients. Two litter-bag decomposition studies were also carried out (i) in 2008 (surface applied) using maize cultivars DKC80-12B, DKC80-10 and DKC6-125 residues and (ii) in 2009 (soil incorporated) using DKC75-15B, CRN3505, PAN6Q-321B v and PAN6Q-121. Ash-free dry matter and Cry1Ab protein were measured throughout the incubation time. There were no differences between DKC61-25B and PAN6Q-321B in terms of expression of Cry1Ab in leaves, stems and roots over time. The Cry1Ab expression levels were in decreasing order: leaves > stems > roots. No effects of Bt maize on earthworm numbers and biomass were observed. There were no differences in all silages parameters except NDF and ADF, which were higher in the Bt maize silage than that of the non-Bt maize from the 2008/09 season. The Cry1Ab levels were essentially not reduced during ensiling. The maize residues (both Bt and non-Bt maize) degraded to similar levels, either when surface-applied or incorporated into soil but soil-incorporated residues decomposed faster than surface-applied ones. Cry1Ab degraded as the plant matrix decomposed. The findings suggested that maize genetically modified with the Bt MON810 event can be grown in the Central Eastern Cape without affecting earthworm numbers and biomass, silage quality and decomposition of maize residues.

The diptericidal $\textit{Bacillus thuringiensis}$ (Bt) ssp. $\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\textit{am1}$ and $\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\delta$-endotoxins (Cry19A from Bt ssp. $\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\textit{240A}$ from Bti and an insertion sequence associated with the $\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\alpha$/$\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\alpha$/$\beta$-type SASP from $\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\textit{Bacilli}$.

Entre os insetos que prejudicam o desenvolvimento sócio-econômico na Serra Gaúcha, encontram-se os dípteros da família Simuliidae, que devido a hematofagia, provocam desconforto e alergias, interferindo no turismo rural. Na área agrícola, os lepidópteros da família Noctuidae, são pragas de diversas culturas, sendo economicamente relevantes. Visando reduzir o uso de substâncias químicas que normalmente são empregadas no controle destes insetos, este trabalho objetivou obter isolados de Bacillus thuringiensis (Bt) para a aplicação em programas regionais de controle de simulídeos e noctuídeos. Utilizando-se amostras de solo da área rural e urbana e de insetos, coletados em municípios da Região, obtiveram-se 721 colônias bacterianas, das quais, 231 (32,03%) foram identificadas como Bt. Testes de patogenicidade contra Culex quinquefasciatus e Anticarsia gemmatalis com 84 isolados de solo e 28 de insetos selecionados por apresentarem elevada taxa de esporulação, indicaram que os recuperados de insetos foram mais específicos, enquanto que maior número dos provenientes de solo foram tóxicos para ambas as espécies, indicando mais amplo espectro entomopatogênico. Apenas um dos 12 isolados apresentou valor de CL50 similar àquele obtido com a linhagem padrão IPS-82, em relação a mortalidade de larvas de C. quinquefasciatus. O perfil das protoxinas dos isolados foi caracterizado por SDS-PAGE, demonstrando que dois isolados obtidos de díptero, tiveram um perfil semelhante a linhagem IPS-82, enquanto dois isolados de solo, com bandas entre 65 e 130 kDa, foram mais tóxicos para lepidópteros e dois obtidos de solo e três de insetos, apresentaram uma banda de aproximadamente 120 kDa, sendo mais efetivos contra lagartas. / Among the insects that harm the socioeconomic development of Serra Gaúcha, exist the dipterous, of the family Simuliidae that, due to its hematofagic process, cause discomfort and allergies to active rural tourists. In the agricultural regions, the lepidopterous, of the family Noctuidae, constitute curses of several cultures, constituting an economical prejudice. Seeking to reduce the use of chemical substances in the control of insects, this work is aimed at isolating the bacteria Bacillus thuringiensis (Bt) for the application of regional programs of Simulids and Noctuids pest control. Samples of insects and of soil of rural and urban areas were collected in the municipal districts of the specific area. Were obtained 721 bacterial colonies, from which, 231 (32,03%) were identified as Bt. Patogenicity tests against Culex quinquefasciatus and Anticarsia gemmatalis were executed with 84 bacteria isolated from soil and 28 bacteria isolated from insects that had a larger capacity of cellular reproduction. The results indicated that the isolated bacteria recovered from the insects were more specific, considering that a larger number of bacteria coming from the soil were more toxic for both species, showing a wider spectrum of entomopatogenic. Just one of the isolated 12 bacteria presented a similar value of CL50 than the one obtained with the lineage pattern IPS-82, in relation of the mortality rate of larvae of C. quinquefasciatus. The profile of the protoxins of one of the isolated ones is characterized by SDS-PAGE, demonstrating that two isolated bacteria obtained from the dipteran, had a similar profile as lineage IPS-82, while two others isolated from the soil, with bands between 65 and 130 kDa, were more toxics for lepidopteran and the two obtained from the soil and three obtained from of insects, and presented one band of approximately 120 kDa, being more effective against caterpillars.

Bacillus cereus is a ubiquitous organism and a potential foodborne pathogen that can cause two types of gastrointestinal diseases: emesis and diarrhea. The emetic syndrome is caused by a heat and acid stable peptide toxin that is pre-formed in food, while the diarrheal syndrome is associated to two 3-protein, heat labile enterotoxin complexes that are formed in the intestine after ingestion of the organism. There are many reports on the isolation and characterization of Bacillus cereus from various foods, however there are no studies on the levels, toxigenicity and physical characteristics of B. cereus isolated from U.S. retail spices. A huge part of spices sold in the U.S. are imported from developing nations. Developing nations lack hygienic practices during processing and packaging of spices, due to which there is a high chance of imported spices being contaminated with B. cereus. Therefore, the main objective of this thesis work was to characterize B. cereus spores from U.S. retail spices. Levels of aerobic spores and B. cereus spores were determined. B. cereus spores were further analyzed for their enterotoxigenic ability, growth characteristics and physical spore characteristics. In the 247 spice samples analyzed 77 were found to contain B. cereus, while 11 were positive for B. thuringiensis. Eighty four of the 88 spices tested possessed either one of the enterotoxin genes. None of the isolates tested positive for the emetic toxin (ces) gene. Seventy five of the B. cereus isolates grew at 12 °C, although only two isolates grew well at 9 °C. Seven selected diarrheal B. cereus spore strains had D95-values ranging from 0.64-3.53 min while the two emetic strains had D95-values of 7.04 min and 6.64 min. B. cereus grew well in pre-cooked rice. After 48 h, counts of 1.26 X 107 and 3.8 X 107 B. cereus/ 10 g were obtained in pre-cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 104 CFU/ 10g even after 48 h of incubation. The aerobic mesophilic bacterial population and B. cereus population of 0.1% crushed pepper in pre-cooked rice over a period of 48h at temperature 20 °C and 17 °C were also analyzed. Counts of B. cereus in pepper rice samples reached a maximum of 1600 MPN/ 10 g and 1100 MPN/ 10 g at 20 °C and 17 °C respectively while the aerobic mesophilic counts per 10 g were 2.4 X 108 and 4.4 X 106 at these temperatures. The low B. cereus counts and high aerobic mesophilic population indicates competition of nutrients in cooked rice by background flora other than B. cereus. The physical spore characteristics of five B. cereus and 3 B. thuringiensis strains were studied using transmission electron microscopy (TEM). Tubular, whip-like appendages were present in four B. cereus and two B. thuringiensis, while all seven isolates possessed exosporia.

The reciprocal evolutionary effects pathogens and their hosts have on each other are one of the most powerful selective forces in evolution, leading to adaptive phenotypic and genetic changes of both antagonists. In nature, bacterial infections often consist of more than one genotype. Since the host represents a limited resource, an interaction between the co-infecting genotypes is likely and potentially has fundamental effects on the interaction with the host. Nevertheless, most studies focus only on the interaction of parasite and host, ignoring within-host dynamics between co-infecting parasite genotypes. In my thesis, I focussed on both, the consequences of long-term host-parasite evolution for the interaction with a host and for parasite-parasite interactions. The first chapter is a comprehensive theoretical overview presenting the effects of multiple infections on virulence towards the host. It summarizes not only potential social interactions between the different co-infecting genotypes, but also discusses the relevance of their relatedness and resulting consequences for virulence. In the second chapter I present the results from a long-term evolution experiment using Caenorhabditis elegans as a multicellular host, singly infected with one of two different strains of its microparasite Bacillus thuringiensis. I found that both, coevolution with and adaptation to the host, led to rapid diversification of the clonal parasite populations into distinct clones. These clones showed strain specific phenotypic changes (i.e., killing rate and production of antagonistic substances) not only within, but also between replicate populations. In the third chapter one of these evolved clones was compared to its ancestral, non-evolved clone on the molecular level. By using next generation genome sequencing, I analysed the underlying genetic mechanisms that led to diversification within the clonal population presented in chapter two. In this study I demonstrated the importance of bacterial genomic plasticity for adaptation: the results revealed that changes were mainly caused by mobile genetic elements (MGEs), especially transposases and plasmids. Overall this thesis shows that the evolutionary selection pressure mediated by a multicellular host causes phenotypic diversification of the parasite. This change within and between parasite populations is reflected on both, the phenotypic and the genetic level.

Transgenic corn hybrids expressing Bt toxins are widely deployed to control pests such as Helicoverpa zea (Boddie). However, Bt resistance can reduce the efficacy of Bt corn hybrids. Volunteer corn expressing Bt toxins may impact Bt resistance management of pests. Surveys in Mississippi revealed the occurrence of fall season volunteer corn in high densities. Helicoverpa zea larvae were found feeding during both vegetative and reproductive stages. However, Bt parentage and low to moderate water and nitrogen stresses did not significantly influence H. zea larval growth and development on Bt positive volunteer plants. Additionally, pollen mediated Bt gene flow to non-Bt refuge ears from Bt hybrids significantly reduced H. zea larval growth on cross-pollinated refuge ears. The implications of these findings are that volunteer corn in most Mississippi corn production regions will have little impact on H. zea Bt resistance management, but resistance management could be compromised in more southern regions.

No description available.

Media for the selective recovery of the mosquito pathogens Bacillus sphaericus 1593 and B. thuringiensis serovar. israelensis were developed. Streptomycin at 100 µg/ml and 500 µg/ml in NY agar (nutrient agar with 0.05% yeast extract) successfully selected for B. sphaericus, and allowed counts equivalent to those obtained on the nutrient medium NY without antibiotics. The medium containing 100 µg/ml of streptomycin (NYST) was used to recover B. sphaericus 1593 from a mixed microbial population in pond water. Sodium chloride, penicillin G, and pH adjustment of the medium were found to be unsatisfactory selective agents. Two selective media for the recovery of B. thuringiensis serovar. israelensis gave counts equivalent to those obtained on the nonselective NY medium. One medium contained 100 µg/ml of polymyxin with 1.0 µg/ml chloramphenicol (NYPC), and the other contained 500 µg/ml of polymyxin alone. The use of the higher level of polymyxin with chloramphenicol reduced the number of viable B. thuringiensis serovar. israelensis. NYPC was used to recover B. thuringiensis serovar. israelensis from a mixed microbial population in pond water. The selective media reduced the number of pond water microorganisms on plates by 90 to 99%. A heat treatment of 50ºC for 10 minutes also reduced pond water microbiota by approximately one log. The use of heat treatment plus either NYST or NYPC reduced the pond water microbiota further. The heat treatment had little effect on sediment microbiota. A selective-differential medium for B. thuringiensis serovar. israelensis was developed for use when heat treatment of samples would be undesirable. This medium, PEMBAC, permitted the observation of peptone deamination and hydrolysis of lecithin, which are characteristic of B. thuringiensis serovar. israelensis. The medium contained 50 µg/ml of polymyxin and 1.0 µg/ml chloramphenicol as selective agents. The parasporal crystals of B. thuringiensis serovar. israelensis are the site of the mosquito larval toxin. Because the crystals are not viable, another method for their enumeration was examined. Antisera to whole crystals and to solubilized crystal antigens were prepared in rabbits for use in the indirect fluorescent antibody technique. Because of the small size and irregular shape of the parasporal crystals of B. thuringiensis serovar. israelensis, the crystals were difficult to distinguish from other small fluorescing particles. The antisera prepared precipitated several antigens in solubilized crystals, but did not adsorb to the majority of the antigens in whole crystals. / Master of Science