Isolation, morphological and molecular characterization of Bacillus thuringiensis strains against Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae)

摘要

The coffee berry borerHypothenemus hampeiFerrari, 1876 (Coleoptera: Curculionidae: Scolytinae) is considered the most serious pest of the coffee crop and is controlled primarily with the use of chemical insecticides. An alternative to this control method is the use of the entomopathogenic bacterium,Bacillus thuringiensisBerliner, 1911. Therefore, the objective of this work was to select strains ofB. thuringiensisvirulent againstH. hampeiand characterize them by morphological and molecular methods to identify possible genes for the production of genetically modified plants. To achieve this objective, 34 strains ofB. thuringiensisunderwent a selective bioassay to evaluate their toxicity toH. hampeifirst-instar larvae. Among the strains tested, 11 and the standardB. thuringiensissubspeciesisraelensis(IPS-82) caused mortality above 90%. Then, the median lethal concentration (LC50) was estimated for these strains followed by characterization using morphological, biochemical and molecular methods. The lowest LC50was obtained for strain BR58, although this concentration did not differ significantly from that of the standard strain IPS-82 or from that of strains BR137, BR80 and BR67. The molecular characterization detectedcry4A,cry4B,cry10,cry11andcyt1genes in 10 of the most virulent strains (BR58, BR137, BR80, BR81, BR147, BR135, BR146, BR138, BR139, BR140). Strain BR67 differed completely from the others and amplified only thecry3gene. This strain was more virulent than BR135, BR146, BR138, BR139 and BR140, but it did not differ from BR58, BR137, BR80, BR81 and BR147. The protein profile revealed proteins of 28, 65, 70 and 130kDa, and the morphological analysis identified spherical crystalline inclusions in all strains. The results showed that the 11 strains studied have potential for use as a gene source for insertion into coffee plants for the controlH. hampei, especially thecry3,cry4A,cry4B,cry10,cry11andcyt1genes, that were repeated in the most virulent isolates.