Multifunctional analysis of new Bacillus thuringiensis strains and their potential as pest control agents

The use of chemical pesticides has been the most extended practice for the control of pests in agriculture for the past 80 years. However, these types of insecticides have experienced strong regulatory measures that limit their use due to their lack of biosecurity. Therefore, the need for safer, sustainable, and environmentally friendly alternatives has never been greater. Bioinsecticides, and more specifically, Bacillus thuringiensis (Bt) based solutions have gained popularity in Integrated Pest Management (IPM) programs due to its biocidal properties, scalability, and overall safety. Bt is a Gram-positive bacterium that forms a resistance spore during the stationary phase of growth and a parasporal crystal which is comprised of d-endotoxins (among others, Cry, and Cyt proteins) with insecticidal activity. Bt is the most commonly applied entomopathogenic agent for pest control in the field and its use is compatible with other control methods. The main aim of this thesis was the study of the strain diversity and pesticidal potential of strains from three Bt libraries. Each of the libraries comprised strains collected from a specific habitat, namely, poultry farms located in the south of the Pyrenees, the meadows of Extremadura, and non-cultivated areas from diverse regions of the world. A total of 294 strains were isolated and clustered into 44 groups or profiles based on their pesticidal gene content. All three habitats showed a moderate pesticidal gene diversity and no direct correlation between specific genes and habitats was found. However some genes were more frequent in certain environments. For instance, although cry1, cry2, and cry9 genes were present in all three habitats, cry1 and cry2 were more relatively abundant in non-cultivated areas. The potential pesticidal properties of the 44 different profiles were characterized in silico based on the available literature and found that the most frequent profiles belonged to strains with the potential to control lepidopteran species (harboring cry1, cry2, and cry9 genes) for all the studied habitats. These were followed by potential nematocidal strains (carrying cry5-like and app6-like genes), which were also identified in all the studied habitats. Strains with hypothetical activity against coleopterans (cry3 and cry8 and similar genes) and dipterans (cry4, cry10, cry11, and cyt) were only found in farms and meadows. The in silico characterization was then verified in vivo, by testing a single concentration (10 µg/ml) of spores and crystals of each profile against representative species of the lepidopteran (Spodoptera littoralis and Helicoverpa armigera), dipteran (Aedes albopictus), and coleopteran (Leptinotarsa decemlineata) insect orders. Interestingly, some of the lepidopteran active profiles identified in this study harbored different gene combinations (cry1C, cry1D, cry1E, cry2A, xpp22-like) to those found in the Bt strains of the most widely used commercial products (DiPel® and Xentari®). Bioassays performed against mosquitoes and coleopterans also provided promising results for groups harboring pesticidal genes that were different from those found in the most commonly used Bt solutions (Eg. Vectobac® and Novodor®, respectively). Today, most Bt-based market products are targeted towards the control of the lepidopteran, coleopteran, and dipteran pests. However, other pests of major importance in agriculture are often out of the scope of said solutions. Some examples of this are mites, nematodes and fungi. One of the objectives of this thesis was to address the multifunctional properties of a Bt strain (from the constructed Bt libraries) with the potential to control several pests, including those for which the current Bt solutions offer no coverage. With this in mind, candidate strain BST-122, which presented an interesting pesticidal gene content, was selected. To evaluate the multifunctional potential of BST-122 bioassays were performed against the Colorado potato beetle (L. decemlineata), for which the spores and crystals produced significant mortality. Furthermore, a Cry5-like protein produced by the strain proved toxic against the challenging phytophagous mite Tetranychus urticae when administered in high concentrations. This result could serve as a basis for searching for new Cry5-like proteins with improved activity against mites. When evaluated against the phytoparasitic nematodes Meloidogyne incognita in plant assays, secreted factors of the strain produced a significant reduction of galls in the roots of cucumber plants. Finally, BST-122 produced a considerable growth deceleration in two different serovars of Fusarium oxysporum (lycopersici and meloni) and Verticillium dahliae in vitro. All in all, this thesis provides insights on the diversity of wild-type Bt strains belonging to different habitats and their potential to control different pest organisms. Additionally, the multifunctional properties of a Bt strain are shown, emphasizing the importance of new proteins and secreted factors to expand the use of Bt solutions beyond the confinements of the Lepidopteran, Coleopteran and Dipteran insect orders and include other pest organisms of economic importance.