Caracterização de isolados de Bacillus Thuringiensis com potencial inseticida: abordagens genômica e proteômica

Abstract

Sustainable alternatives that can replace chemical insecticides are necessary for insect pest control. Bacillus thuringiensis (Bt) is a bacterium that produces insecticidal proteins toxic against insect pests of different orders. In this work, we performed bioassays using Bt strains isolated from Tocantins against dipterous and lepidopteran insects. To explore all the genes related to the pathogenicity of the isolates, the genomes of these bacteria were sequenced. Furthermore, in chapters two and four, proteomic was combined with genomic analysis to detect proteins expressed in the spore-crystal mixture. In chapter one, a comparative analysis of four genomes of Bacillus thuringiensis subsp. israelensis strains with toxicity to Aedes aegypti and Culex quinquefasciatus revealed high nucleotide sequence identity (>98%), the same plasmids profile, and equal pesticidal protein content (cry4Ba, cry4Aa, cry11Aa, cry10Aa, cyt1Aa, cyt2Ba, and cytCa). The genome of the Bt TOD651 strain, presented in Chapter 2, with toxic activity to A. aegypti and C. quinquefasciatus (CL50 of 0.011 and 0.023 μg/mL, respectively), showed CDS regions highly homologous to cry11Aa3, cry10Aa4, cry4Aa4, cry4Ba5, cyt1Aa5, cyt1Ca1, cyt2Ba13, mpp60Aa3, and mpp60Ba3 genes. The expression of Cry11Aa3, Cry10Aa4, Cry4Aa4, Cry4Ba5, Cyt1Aa5, Cyt1Ca1, Cyt2Ba13, and Mpp60Ba3 proteins was identified in the spore-crystal mixture, of which Cry4Ba5 was more abundant than Cyt1Aa5. The expression of the enzyme Mppe was the most abundant among the proteases. In chapter 3, the Bt UFT038, tested for different soybean pests, showed higher toxicity to Spodoptera cosmioides (CL50=6.8 106/cm2), and its genomic analysis revealed the presence of cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Finally, in chapter 4, Bt strain TOL651, phylogenetically close to subspecies kenyae, was more toxic to Anticarsia gemmatalis (LC50 =1.45 ng.cm-2) compared to Diatraea saccharalis (LC50 = 73.77 ng.cm-2). Its genomic analysis allowed the detection of cry1Aa18, cry1Ia44, cry2Aa9, and cry1Ac5 genes, while proteomics indicated expression of Cry1Aa18, Cry1Ac5, and Cry2Aa9 proteins, of which Cry1Ac5 was most abundant. In addition, the virulence factor InhA1 was detected and thus should also contribute to the toxicity of this isolate. In conclusion, the Bt isolates of this study are alternatives for biological control, and genomic and genomic- proteomic characterization are important steps that could contribute to the development of new biocontrol strategies for disease vector mosquitoes and agricultural pests.