Complete genome sequence of Bacillus thuringiensis BR145, a strain with insecticidal activity against Lepidoptera pests.

Bacillus thuringiensis BR145 isolated from a soybean field in Southern Brazil showed toxicity against two important insect pests from soybean crop, Helicoverpa armigera, and Chrysodeixis includens, with LC50 0.294 µg.cm-2 and 0.277 µg.cm-2, respectively. We analyzed the genome of this strain through sequences obtained by Next Generation DNA Sequencing and de novo assembly. The analysis of the genome revealed insecticidal genes cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Ab, cyt1, and vip3Aa, suggesting the use of this strain in new strategies of biological control.

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

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.

A New Approach to Chromosomal Evolution in the Giant Water Bug (Heteroptera: Belostomatidae).

The genus Belostoma, known colloquially as "giant water bugs," presents striking cytogenetic diversity and extensive chromosome variability. Notwithstanding, its karyotype evolution is not well understood. We analyzed 8 species of Belostoma (77 samples). The meiotic analysis revealed 2n = 14 + XY for Belostoma horvathi and Belostoma candidulum; 2n = 22 + XY for Belostoma cummings; 2n = 26 + X1X2Y for Belostoma dentatum, Belostoma elongatum, and Belostoma discretum; and 2n = 26 + X1X2X3Y for Belostoma testacopallidum and Belostoma dilatatum. All species showed holokinetic chromosomes. Based on heterochromatin distribution patterns and 18S rDNA, the species of the genus Belostoma were separated into four groups. The analysis of C0t-1 DNA showed that the repetitive DNA, partly composed of microsatellite DNA, was absent on the Y chromosome. Fluorescent in situ hybridization (FISH) using a microdissected X chromosome in species with simple sex system presents uniform hybridization in the nuclear region corresponding to the X chromosome. Species with multiple systems revealed discrete markings. The present data in conjunction with the existing literature led us to propose a new evolutionary hypothesis for the group, with an ancestral karyotype with a low diploid number, simple sex determination system, and nucleolus organizer regions (NORs) on the sex chromosomes. That karyotype would have originated other karyotypes through agmatoploidy, simploidy, heterochromatinization, and movement of the 18S rDNA.

Susceptibility of Grapholita molesta (Busck, 1916) to formulations of Bacillus thuringiensis, individual toxins and their mixtures.

The Oriental fruit moth, Grapholita molesta (Lepidoptera: Tortricidae), is a major pest of fruit trees worldwide, such as peach and apple. Bacillus thuringiensis has been shown to be an efficient alternative to synthetic insecticides in the control of many agricultural pests. The objective of this study was to evaluate the effectiveness of B. thuringiensis individual toxins and their mixtures for the control of G. molesta. Bioassays were performed with Cry1Aa, Cry1Ac, Cry1Ca, Vip3Aa, Vip3Af and Vip3Ca, as well as with the commercial products DiPel® and XenTari®. The most active proteins were Vip3Aa and Cry1Aa, with LC50 values of 1.8 and 7.5ng/cm2, respectively. Vip3Ca was nontoxic to this insect species. Among the commercial products, DiPel® was slightly, but significantly, more toxic than XenTari®, with LC50 values of 13 and 33ng commercial product/cm2, respectively. Since Vip3A and Cry1 proteins are expressed together in some insect-resistant crops, we evaluated possible synergistic or antagonistic interactions among them. The results showed moderate to high antagonism in the combinations of Vip3Aa with Cry1Aa and Cry1Ca.