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.

Genome Sequence of the Mosquitocidal Bacillus thuringiensis Strain BR58, a Biopesticide Product Effective against the Coffee Berry Borer (Hypothenemus hampei).

Bacillus thuringiensis is an important microbial control agent against insect pests. The draft genome sequence of the Brazilian strain BR58 described here contains the insecticidal genes cry4A, cry4B, cry10A, cry11A, cry60A, cry60B, and cyt1A, which show toxicity to both Aedes aegypti and Hypothenemus hampei larvae.

In vitro sensitivity of antagonistic Trichoderma atroviride to herbicides

Trichoderma atroviride was tested in vitro for its sensitivity to different herbicides. The dosages tested were recommended dosage (RD), half dosage (½RD), and double dosage (2RD). Germination, colony-forming units (CFU), radial growth, and spore production were evaluated. Carfentrazone-ethyl and sulfentrazone inhibited the germination at RD and 2RD. A reduction in the CFU was observed for glufosinate-ammonium, atrazine, carfentrazone-ethyl, diuron + paraquat dichloride, imazapyr, oxyfluorfen, and sulfentrazone at each of the tested dosages. Radial growth was influenced by ametryn, atrazine, carfentrazone-ethyl, oxyfluorfen, and sulfentrazone herbicides, with an 80% reduction of the colonial area. Spore production was affected by carfentrazone-ethyl, oxyfluorfen, and sulfentrazone with colonial area reductions of over 70%. It was concluded that 2,4 D, clomazone, and imazapyr herbicides showed the least toxicity to T. atroviride and should be used in the crops where the fungus has been applied for phytopathogen control.

Produção de esporos de Beauveria bassiana (Bals). Vuill. num processo bifásico utilizando diferentes meios líquidos / Beauveria bassiana (Bals.) Vuill. spores production in biphasic process utilizing different liquid media

Técnicas de produção de fungos entomopatogênicos são desenvolvidas buscando aumentar a produtividade desses patógenos e reduzir custos do processo. O objetivo deste trabalho foi avaliar a produção de biomassa de Beauveria bassiana em meios líquidos e a conidiogênese no processo bifásico. Os meios líquidos testados foram: FC (farinha de crisálida), BD (batata+dextrose) e FCBD (farinha de crisálida+batata+dextrose). Os períodos de avaliação da produção de biomassa foram: 24, 48, 72, 96, 120 e 144 hs. Para produção pelo processo bifásico, diferentes quantidades dos meios líquidos (5, 10, 15 e 20 mL) com diferentes períodos de incubação (24, 48 e 72 hs) foram adicionadas sobre arroz pré-cozido (200 g), avaliando-se a produção de conídios após 10 dias. Na produção de biomassa, o meio FCBD foi o mais produtivo durante todos os períodos avaliados. No processo bifásico, as maiores produções de conídios foram atingidas quando utilizou-se como inóculo do arroz, biomassa fúngica produzida nos meios FC e FCBD (2,7 x 10 12 e 2,8 x 10 12 conídios/g de arroz). As quantidades de 5, 15 e 20 mL de meio líquido, inoculadas sobre arroz, nåo diferiram estatisticamente. Os tempos de incubação de 24 e 48 hs, do fungo em meio líquido, proporcionaram maior conidiogênese do que em 72 hs. Os dados mostram a necessidade de fontes de carbono e nitrogênio na produção de biomassa em meio líquido e na conidiogênese em meio sólido. Também, é possível otimizar o processo bifásico, adicionando sobre o arroz (200 g) 5 mL do meio líquido FCBD com 24 hs de incubação.