3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The evaluation of Bacillus thuringiensis subspecies israelensis based biopesticides for the control of southern African malaria vectors(2012) Ketseoglou, IreneMalaria, one of the most deadly vector-borne diseases in the world, is transmitted by the bite of an infected female Anopheles mosquito. Since there are several problems arising from current control programs, alternative control strategies for malaria-carrying vectors are required. The objective of this thesis was to evaluate a recombinant Anabaena sp. strain PCC 7120, Anabaena PCC 7120#11 (PCC 7120#11), as an agent to control southern African malaria vectors. Select Bacillus thuringiensis subspecies israelensis (Bti) toxin genes, cry4Aa and cry11Aa, had previously been cloned into Anabaena sp. strain PCC 7120. Evaluation of the potential of PCC 7120#11 and Bti as control agents of southern African malaria vectors required the evaluation of the larvicidal activity of PCC 7120#11 and Bti against several important malaria vectors. This study showed that all five of the African anopheline species evaluated in laboratory bioassays (Anopheles gambiae, Anopheles arabiensis, Anopheles funestus, Anopheles quadriannulatus, and Anopheles merus) were susceptible to Bti, whereas PCC 7120#11 exhibited high larvicidal activity against four of the five anopheline species. The low larvicidal activity of PCC 7120#11 against An. funestus could be due to the absence of the other Bti toxins in PCC 7120#11 or differences in the structure and/or density of specific midgut toxin receptors for the Cry4Aa or Cry11Aa proteins. The Standardized Aquatic Microcosm (SAM), a synthetic multi-species system consisting of a chemically defined medium with the same species and concentration of photosynthetic microorganisms (PMOs) and invertebrates, was used to comprehensively evaluate the effects of Bti and PCC 7120#11 in an aquatic environment. The SAM experiments showed that Bti was non-toxic to the invertebrates evaluated and did not significantly affect any of the other variables evaluated in the microcosms. Bti was shown to be an environmentally friendly control agent.Item Isolation of a Bacillus thuringiensis strain from South African soils and the characterization of its cry gene sequence.(2007-10-23T13:06:43Z) Laridon, Neil EdwardThe objectives of this project was to isolate and characterize a Bacillus thuringiensis strain from South African soils, determine its cry gene sequence, clone this gene sequence and determine its toxicity. Forty four putative Bacillus thuringiensis strains were extracted from soil samples taken from the Muldersdrift mountain range, the Roodekrans botanical gardens, Southbroom in Kwazulu Natal and Nelspruit in eastern Mpumalanga province. The bacterial populations of these soil samples were isolated and classified using different microbial, and biochemical techniques including sodium acetate tests to isolate putative B. thuringiensis spores. These spores were cultured and further characterized through colony shape and colour as well as the presence of δ-endotoxin crystals. Once characterized, DNA was extracted from the isolates using an array of techniques to obtain high quality template DNA. This DNA was then screened via PCR using truncated versions of the cry1A specific primers TYIAA (f) and TYIUN12 (r). The insecticidal protein CRY1A was selected for this study since it is specific and highly toxic to lepidopteron insects. Homology to the cry1a gene was detected in six of the Bacillus strains analyzed, namely S4, S9, S10 n1, n3 and n5. PCR products were cloned into the pTZ57R/T cloning vector and transformed into JM109 competent cells. DNA from the six isolates was also characterized at the 16S rDNA level with the use of PCR. Primers capable of amplifying nearly full-length 16S ribosomal DNA (approximately l,500-bp) fragments from many bacterial genera confirmed that the isolates were indeed Bacillus thuringiensis, showing evidence of lineage according to the signature sequences within the conserved regions. Spore/δ-endotoxin mixtures of the randomly selected isolate S10 were used in a bioassay to test their toxicity against the lepidopteron insect Galleria mellonella. No significant mortalities were reported, but sensitivity to the S10 δ- endotoxin was evident when compared to results using known B. thuringiensis δ- endotoxins at the same concentrations.