RESUMO
ABSTRACT The coffee berry borer Hypothenemus hampei Ferrari, 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 thuringiensis Berliner, 1911. Therefore, the objective of this work was to select strains of B. thuringiensis virulent against H. hampei and characterize them by morphological and molecular methods to identify possible genes for the production of genetically modified plants. To achieve this objective, 34 strains of B. thuringiensis underwent a selective bioassay to evaluate their toxicity to H. hampei first-instar larvae. Among the strains tested, 11 and the standard B. thuringiensis subspecies israelensis (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 LC50 was 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 detected cry4A, cry4B, cry10, cry11 and cyt1 genes 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 the cry3 gene. 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 130 kDa, 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 control H. hampei, especially the cry3, cry4A, cry4B, cry10, cry11 and cyt1 genes, that were repeated in the most virulent isolates.
RESUMO
Bacillus thuringiensis isolates were obtained from soil samples collected at different sites located in the same region but with different vegetation. The sites showed different frequencies of B. thuringiensis, depending on the type of vegetation. Strains of B. thuringiensis were found to be less common in samples of riparian forest soil than in soil of other types of vegetation. The rate of occurrence of B. thuringiensis in the samples also varied according to the vegetation. These results show that whenever this bacterium was found, it showed a high rate of occurrence, indicating that this species could be better adapted to using soil as a reservoir than other Bacillus species. The presence of cry genes was analyzed by polymerase chain reaction, and genes that exhibited activity against Diptera species were the most commonly found. The isolates obtained were characterized by random amplified polymorphic DNA, and 50% were clustered into clonal groups. These results demonstrated the possible occurrence of a high number of genetically similar strains when samples are collected from the same region, even if they are from locations with different vegetation.
