Characterization, distribution and cloning of Bacillus thuringiensis cry1 genes efficient against fall armyworm (Spodoptera frugiperda) in Brazil

Brazil is located in South America and contains a rich and different biodiversity. A total of 4,459 Bt strains were isolated and evaluated regarding to Spodoptera frugiperda larval mortality, and 165 showed larval mortality above 75%. Molecular characterization was based on PCR electrophoresis profile using specific cry1 primers. Among these strains, 33 (20%) did not amplify the expected fragments; 103 (62,42%) amplified fragments corresponding to the presence of only one gene, while 25 (15,15%), 3 (1,8%) and 1 (0,6%) showed a profile of two, three and four different cry1 genes, respectively. SDS-page protein analyses were positive for the presence of cry1 genes. The most frequent (57.5%) was cry1D gene, whereas cry1Aa/cry1Ad and cry1C genes were the less frequent (1.2%). However, more than 60% of the evaluated strains presented cry1B and cry1E genes. Analysis of strains carrying cry1C, cry1B, cry1E, cry1F, cry1A, cry1G and cry1D genes showed that they were toxic S. frugiperda, ranging from the most to the least toxic. The available sequences at http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ were used for designing primers to clone cry1C and cry1F genes. The amplified fragments with the expected size, approximately 2,046 bp, were purified, cloned and transformed into competent cells. The sequencing of 5' and 3' ends allowed the confirmation of the identity of the genes. Some strains that presented unspecific fragments, were also cloned, amplified, sequenced and showed sequences corresponding to cry1-type genes. Colonies holding clones of cry1F, cry1Ca and cry1Cb genes, were obtained only for two of the evaluated strains.