RESUMEN
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.
RESUMEN
Bovine hemoplasmas are bacteria found on the erythrocyte surface or free in the plasma of cattle. The aim of the present study was to evaluate the occurrence of 'Candidatus Mycoplasma haemobos' ('C. M. haemobos') in Holstein and Jersey cattle raised in Londrina and surroundings, northern region of the State of Parana, Southern Brazil. PCR testing directed to 16S rRNA gene fragment was performed to investigate the occurrence and characterize the molecular identity of 'C. M. haemobos'. A total of 264/433 (60.97%) blood samples were positive by PCR. Further alignment of 500-bp amplicons to available sequences at the GenBank database showed high identity (100%) to 'C. M. haemobos'. To the author's knowledge, this is the first molecular confirmation of the hemoplasma 'C. M. haemobos' in cattle from Brazil. Moreover, 'C. M. haemobos' was observed in high occurrence in dairy cattle, and may have significant impact in livestock production.
Hemoplasmas de bovinos são bactérias encontradas na superfície de hemácias, ou livre no plasma de bovinos. O objetivo do presente estudo foi avaliar a ocorrência de Candidatus Mycoplasma haemobos' ('C. M. haemobos') em bovinos das raças Holandesa e Jersey da região de Londrina, norte do Paraná, sul do Brasil. Para investigar a ocorrência e caracterizar a identidade molecular do 'C. M haemobos' uma PCR baseada no fragmento do gene 16S rRNA foi realizada. A PCR identificou como positivas 264/433 (61%) amostras de sangue testadas. O alinhamento deste fragmento de 500 pb com seqüências disponíveis no GenBank mostrou 100% de identidade 'C. M. haemobos'. Pela bibliografia consultada, esta é a primeira confirmação molecular do hemoplasma 'C. M. haemobos' em bovinos no Brasil. Além disso, foi observada uma alta prevalência deste hemoplasma em bovinos de leite, que pode ter um impacto importante na pecuária bovina.