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. Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae) é considerada praga para mais de 60 espécies de plantas cultivadas como o milho, cana-de-açúcar, trigo, soja, arroz, feijão, sorgo, amendoim e algodão. O objetivo deste trabalho foi selecionar e caracterizar isolados de Bacillus thuringiensis virulentos a E. lignosellus atuando como método alternativo de controle. Assim, 47 isolados foram avaliados em bioensaios seletivos contra lagartas de 1º ínstar de E. lignosellus. Para os isolados mais tóxicos, a concentração letal (CL50) foi estimada e a sua caracterização foi realizada por métodos morfológicos, bioquímicos e moleculares. Dos 47 isolados testados, 12 causaram mortalidade acima 85% e apresentaram CL50 entre 0.038E+8 a 0.855E+8 esporos mL-1. Os menores valores de CL50 foram obtidos pelos isolados BR83, BR145, BR09, BR78, S1534 e S1302, os quais não diferiram da linhagem padrão HD-1, com exceção do BR83. O perfil proteico revelou proteínas Cry entre 60 e 130 kDa, e a caracterização molecular mostrou a presença dos genes cry1, cry2, cry3 e cry11. A análise morfológica identificou três diferentes inclusões cristalinas: bipiramidais, esféricas e cuboides. Entre os isolados avaliados, 12 apresentam potencial biotecnológico para controle de E. lignosellus via formulação de novos bioinseticida ou produção de plantas transgênicas.

ABSTRACT. Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae) is an insect pest of 60 economically important crops, including sugarcane, wheat, soybean, rice, beans, sorghum, peanuts, and cotton. The aim of this work was to select and characterize Bacillus thuringiensis isolates with insecticidal activity against E. Lignosellus that could be used as an alternative method of control. Selective bioassays were done to evaluate the toxicity of 47 isolates against first instar larvae of E. lignosellus. For the most toxic bacterial strains, the lethal concentration (LC50) was estimated and morphological, biochemical and molecular methods were used to characterize the isolates. Among the 47 isolates tested, 12 caused mortality above 85% and showed LC50 values from 0.038E+8 to 0.855E+8 spores mL-1. Isolates BR83, BR145, BR09, BR78, S1534, and S1302 had the lowest LC50 values and did not differ from the standard HD-1 strain; the exception was BR83.The protein profiles produced bands with molecular masses of 60-130 kDa. The genes cry1, cry2, cry3, and cry11 were identified in the molecular characterization. The morphological analysis identified three different crystal inclusions: bipyramidal, spherical and cuboidal. Among the tested isolates, 12 isolates have potential for biotechnological control of E. Lignosellus by development of new biopesticides or genetically modified plants.

Resumo:O objetivo deste trabalho foi selecionar e caracterizar molecularmente isolados de Bacillus thuringiensistóxicos a Spodoptera eridaniaeS. frugiperda. Trinta e quatro isolados foram submetidos ao bioensaio, dos quais três foram selecionados e usados para a estimativa da CL50. Os isolados selecionados não diferiram da linhagem padrão HD-1. Na caracterização molecular, identificou-se a presença dos genes cry1 e cry2, nos isolados BR37 e BR94, e dos genes cry4A, cry4B, cry10, cry11 e cyt1 no isolado BR58, o que confirmou o perfil proteico obtido de 130, 70 e 65 kDa. Foram identificados cristais bipiramidais e esféricos. O isolado BR58, apesar de não conter os genes relacionados à toxicidade a Lepidoptera, causa mortalidade em ambas as espécies

Abstract:The objective of this work was to select and molecularly characterize Bacillus thuringiensis isolates toxic to Spodoptera eridaniaand S. frugiperda. Thirty four isolates were subjected to the bioassay, of which three were selected and used to estimate the LC50. The selected isolates did not differ from the HD-1 standard line. In the molecular characterization, the presence of the cry1 and cry2 genes was identified in the BR37 and BR94 isolates, and of the cry4A, cry4B, cry10, cry11, and cyt1 genes in the BR58 isolate, which confirmed the obtained protein profile of 130, 70, and 65 kDa. Bipyramidal and spherical crystals were identified. The BR58 isolate, although not containing the genes related to toxicity to Lepidoptera, causes mortality in both species.