Selection and characterization of Bacillus thuringiensis efficient strains against Diatraea saccharalis (Lepidoptera: Crambidae)

O objetivo deste trabalho foi selecionar e caracterizar estirpes nativas de Bacillus thuringiensis tóxicas a Diatraea saccharalis (Lepidoptera: Crambidae). Cento e seis estirpes pertencentes ao Banco de Bactérias de Invertebrados, da Embrapa Recursos Genéticos e Biotecnologia, foram testadas quanto à toxicidade a D. saccharalis, e, as mais tóxicas, caracterizadas por métodos bioquímicos e moleculares. Das 106 estirpes testadas, 16 causaram 100% de mortalidade em 24 horas. As três estirpes mais tóxicas apresentaram concentração letal média entre 8 e 43 ng cm-2. O perfil proteico das 16 estirpes mostrou a presença de proteínas de 130 e 65 kDa, e a caracterização molecular mostrou a presença dos genes tipo cry1 e cry2: cry1Aa, cry1Ab, cry1Ac e cry2Aa. A concentração letal média de esporos e cristais obtidos a partir de estirpes recombinantes, que expressavam individualmente os genes cry1Aa, cry1Ab, cry1Ac e cry2Aa, variou entre 222 e 610 ng cm-2, valores muito superiores aos das estirpes nativas mais tóxicas, que apresentavam possibilidade de expressão simultânea desses genes. Este resultado é indicativo de que há sinergia entre as toxinas. Há interação entre as toxinas de B. thuringiensis e seus receptores na broca-do-colmo da cana-de-açúcar.The objective of this work was to select and characterize native strains of Bacillus thuringiensis toxic to Diatraea saccharalis (Lepidoptera: Crambidae). A hundred-and-six strains, belonging to the bank of invertebrate bacteria (Brazil), of Embrapa Genetic Resources and Biotechnology, were tested as to their toxicity to D. saccharalis, and the most toxic ones were characterized by biochemical and molecular methods. Out of the 106 tested strains, 16 caused 100% mortality within 24 hours. The three most toxic strains showed median lethal concentrations between 8 and 43 ng cm-2. The protein profile of the 16 strains showed the presence of 130 and 65 kDa proteins, and the molecular characterization showed the presence of cry1and cry2 type genes: cry1Aa, cry1Ab, cry1Ac, and cry2Aa. The median lethal concentration of spores and crystals obtained from recombinant strains, which individually expressed the genes cry1Aa, cry1Ab, cry1Ac, and cry2Aa, varied between 222 and 610 ng cm-2, values much higher than the ones of the most toxic native strains, which have the possibility of simultaneously expressing these genes. This result indicates a synergy between the toxins. There are interactions between B. thuringiensis toxins and their receptors in the sugarcane borer

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

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Seleção e caracterização de estirpes de Bacillus thuringiensis eficientes contra a Diatraea saccharalis (Lepidoptera: Crambidae)

O objetivo deste trabalho foi selecionar e caracterizar estirpes nativas de Bacillus thuringiensis tóxicas a Diatraea saccharalis (Lepidoptera: Crambidae). Cento e seis estirpes pertencentes ao Banco de Bactérias de Invertebrados, da Embrapa Recursos Genéticos e Biotecnologia, foram testadas quanto à toxicidade a D. saccharalis, e, as mais tóxicas, caracterizadas por métodos bioquímicos e moleculares. Das 106 estirpes testadas, 16 causaram 100% de mortalidade em 24 horas. As três estirpes mais tóxicas apresentaram concentração letal média entre 8 e 43 ng cm-2. O perfil proteico das 16 estirpes mostrou a presença de proteínas de 130 e 65 kDa, e a caracterização molecular mostrou a presença dos genes tipo cry1 e cry2: cry1Aa, cry1Ab, cry1Ac e cry2Aa. A concentração letal média de esporos e cristais obtidos a partir de estirpes recombinantes, que expressavam individualmente os genes cry1Aa, cry1Ab, cry1Ac e cry2Aa, variou entre 222 e 610 ng cm-2, valores muito superiores aos das estirpes nativas mais tóxicas, que apresentavam possibilidade de expressão simultânea desses genes. Este resultado é indicativo de que há sinergia entre as toxinas. Há interação entre as toxinas de B. thuringiensis e seus receptores na broca-do-colmo da cana-de-açúcar

Seleção e caracterização de estirpes de Bacillus thuringiensis eficientes contra a Diatraea saccharalis (Lepidoptera: Crambidae) Selection and characterization of Bacillus thuringiensis efficient strains against Diatraea saccharalis (Lepidoptera: Crambidae)

O objetivo deste trabalho foi selecionar e caracterizar estirpes nativas de Bacillus thuringiensis tóxicas a Diatraea saccharalis (Lepidoptera: Crambidae). Cento e seis estirpes pertencentes ao Banco de Bactérias de Invertebrados, da Embrapa Recursos Genéticos e Biotecnologia, foram testadas quanto à toxicidade a D. saccharalis, e, as mais tóxicas, caracterizadas por métodos bioquímicos e moleculares. Das 106 estirpes testadas, 16 causaram 100% de mortalidade em 24 horas. As três estirpes mais tóxicas apresentaram concentração letal média entre 8 e 43 ng cm-2. O perfil proteico das 16 estirpes mostrou a presença de proteínas de 130 e 65 kDa, e a caracterização molecular mostrou a presença dos genes tipo cry1 e cry2: cry1Aa, cry1Ab, cry1Ac e cry2Aa. A concentração letal média de esporos e cristais obtidos a partir de estirpes recombinantes, que expressavam individualmente os genes cry1Aa, cry1Ab, cry1Ac e cry2Aa, variou entre 222 e 610 ng cm-2, valores muito superiores aos das estirpes nativas mais tóxicas, que apresentavam possibilidade de expressão simultânea desses genes. Este resultado é indicativo de que há sinergia entre as toxinas. Há interação entre as toxinas de B. thuringiensis e seus receptores na broca-do-colmo da cana-de-açúcar.The objective of this work was to select and characterize native strains of Bacillus thuringiensis toxic to Diatraea saccharalis (Lepidoptera: Crambidae). A hundred-and-six strains, belonging to the bank of invertebrate bacteria (Brazil), of Embrapa Genetic Resources and Biotechnology, were tested as to their toxicity to D. saccharalis, and the most toxic ones were characterized by biochemical and molecular methods. Out of the 106 tested strains, 16 caused 100% mortality within 24 hours. The three most toxic strains showed median lethal concentrations between 8 and 43 ng cm-2. The protein profile of the 16 strains showed the presence of 130 and 65 kDa proteins, and the molecular characterization showed the presence of cry1and cry2 type genes: cry1Aa, cry1Ab, cry1Ac, and cry2Aa. The median lethal concentration of spores and crystals obtained from recombinant strains, which individually expressed the genes cry1Aa, cry1Ab, cry1Ac, and cry2Aa, varied between 222 and 610 ng cm-2, values much higher than the ones of the most toxic native strains, which have the possibility of simultaneously expressing these genes. This result indicates a synergy between the toxins. There are interactions between B. thuringiensis toxins and their receptors in the sugarcane borer

Método e composições para controle genético de insetos-praga em plantas de algodão através do silenciamento de genes da família da lacase

Universidade Federal do Rio Grande do SulEmpresa Brasileira de Pesquisa AgropecuáriaDepositad

Método e composições para controle genético de insetos-praga em plantas de algodão através do silenciamento de genes da família da lacase

Universidade Federal do Rio Grande do SulEmpresa Brasileira de Pesquisa AgropecuáriaDepositad

Knocking-down Meloidogyne incognita proteases by plant-delivered dsRNA has negative pleiotropic effect on nematode vigor

The root-knot nematode Meloidogyne incognita causes serious damage and yield losses in numerous important crops worldwide. Analysis of the M. incognita genome revealed a vast number of proteases belonging to five different catalytic classes. Several reports indicate that M. incognita proteases could play important roles in nematode parasitism, besides their function in ordinary digestion of giant cell contents for feeding. The precise roles of these proteins during parasitism however are still unknown, making them interesting targets for gene silencing to address protein function. In this study we have knocked-down an aspartic (Mi-asp-1), a serine (Mi-ser-1) and a cysteine protease (Mi-cpl-1) by RNAi interference to get an insight into the function of these enzymes during a host/nematode interaction. Tobacco lines expressing dsRNA for Mi-ser-1 (dsSER), Mi-cpl-1 (dsCPL) and for the three genes together (dsFusion) were generated. Histological analysis of galls did not show clear differences in giant cell morphology. Interestingly, nematodes that infected plants expressing dsRNA for proteases produced a reduced number of eggs. In addition, nematode progeny matured in dsSER plants had reduced success in egg hatching, while progeny resulting from dsCPL and dsFusion plants were less successful to infect wild-type host plants. Quantitative PCR analysis confirmed a reduction in transcripts for Mi-cpl-1 and Mi-ser-1 proteases. Our results indicate that these proteases are possibly involved in different processes throughout nematode development, like nutrition, reproduction and embryogenesis. A better understanding of nematode proteases and their possible role during a plant-nematode interaction might help to develop new tools for phytonematode control

The Mi-EFF1/Minc17998 effector interacts with the soybean GmHub6 protein to promote host plant parasitism by Meloidogyne incognita

Background: Meloidogyne incognita is the most frequently reported species from the root-knot nematode (RKN) complex responsible for causing damage in several different crops worldwide. The interaction between M. incognita and host plants involves the secretions of molecular factors from the nematode, which mainly suppress the defense response and promote plant parasitism. On the other hand, several plant elements are associated with the immune defense system that opposes nematode infection. Results: In this study, the interaction of the Mi-EFF1/Minc17998 effector with the soybean GmHub6 (Glyma.17G099100; TCP14) protein was identified and characterized in vitro and in vivo . Data showed that the GmHub6 gene is upregulated by M. incognita infection in a nematode-resistant soybean cultivar (PI595099) compared to a susceptible cultivar (BRS133). Accordingly, the Arabidopsis thaliana AtHub6 mutant line (AT3G47620, orthologous gene of GmHub6 displayed normal vegetative development of the plant but was more susceptible to M. incognita . Thus, since the soybean and A. thaliana Hub6 proteins are TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors involved in plant development and morphogenesis modulation, flowering time regulation, and the activation of the plant immune system, our data suggest that the interaction of Mi-EFF1/Minc17998 and Hub6 proteins is associated with an increase in plant susceptibility to nematode infection during parasitism. It is suggested that this interaction may prevent the nuclear localization or disturb the activity of GmHub6 as a typical transcription factor modulating the cell cycle of the plant, avoid the activation of the host’s defense response, and successfully promote parasitism. Conclusion: Our findings indicate the potential of the Mi-EFF1/Minc17998 effector for the development of biotechnological tools based on the approaches of RNA interference and GmHub6 gene overexpression for RKN control

Primer sequences.

<p>Primer sequences.</p