RESUMO
Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-¹ combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-¹ level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-¹ to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.(AU)
A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-¹, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-¹ em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-¹ como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-¹ para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.(AU)
Assuntos
Gossypium/genética , Gossypium , Bacillus thuringiensis/genética , Controle de Pragas/métodos , Nitrogênio/administração & dosagem , Fósforo/administração & dosagem , Fertilizantes/análise , Ensaio de Imunoadsorção Enzimática , Reação em Cadeia da PolimeraseRESUMO
Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.
Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.
Assuntos
Animais , Proteínas Hemolisinas/genética , Mariposas , Fósforo , Proteínas de Bactérias/genética , Resistência a Inseticidas , Plantas Geneticamente Modificadas/genética , Endotoxinas/genética , Fertilizantes , Toxinas de Bacillus thuringiensis , Larva , NitrogênioRESUMO
Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-¹ combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-¹ level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-¹ to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.
A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-¹, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-¹ em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-¹ como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-¹ para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.
Assuntos
Bacillus thuringiensis/genética , Controle de Pragas/métodos , Fertilizantes/análise , Fósforo/administração & dosagem , Gossypium , Gossypium/genética , Nitrogênio/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Reação em Cadeia da PolimeraseRESUMO
Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance ( 1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.
Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração ( 1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.
RESUMO
Cultivated cotton (Gossypium hirsutum) is heavily attacked by various species of insects worldwide and breeding of new varieties resistant to pests is still a hard battle to win. RNAi technology is an important reverse genetics tool to induce gene silencing in eukaryotic organisms and produce phenotypic modifications. In cotton, RNAi was applied to investigate gene function and enhance resistance to insects and pathogens. Different methods and techniques can be used to synthetize double stranded RNA (dsRNA) into plant cells. The Agrobacterium-mediated transformation is a common method to introduce RNAi binary plasmids into cotton genome and obtain stable transgenics plants. This methodology includes the coculture of cotton tissues with Agrobacterium cultures, selection of transgenic cells and induction of somatic embryogenesis to finally obtain transgenic plants after a relatively long period of time. The transient synthesis of dsRNA mediated by virus-induced gene silencing (VIGS) in cotton is an alternative to anticipate the silencing effect of a specific RNA sequence, prior to the development of a stable transgenic plant. VIGS vectors are incorporated into the plant by agroinfiltration technique. During VIGS replication inside plant cells, synthetized dsRNA allows the study on specific heterologous gene expression including the phenotypic effect on herbivorous target pests, thus facilitating a rapid evaluation of dsRNA expressed in cotton plants against individual insect target genes. Here we describe the complementation of these two techniques to evaluate RNAi-based cotton plant protection against insect pests.
Assuntos
Gossypium , Agrobacterium/genética , Animais , Gossypium/genética , Insetos , Melhoramento Vegetal , Plantas Geneticamente Modificadas/genética , Interferência de RNA , RNA de Cadeia Dupla/genéticaRESUMO
ABSTRACT The host acceptance behavior and environmental factors as temperature affect the feeding behavior of Lepidoptera pests. Thus, they must be considered in studies about the risk potential of resistance evolution. The current study sets the differences in the feeding behavior of neonate Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) larvae exposed to Bt and non-Bt cotton plants, under different temperatures and time gap after hatching. Two cotton cultivars were used: the Bt (DP 404 BG - bollgard) and the non-transformed isoline, DP 4049. We found that the feeding behavior of neonate A. argillacea is significantly different between Bt and non-Bt cotton. Based on the number of larvae with vegetal tissue in their gut found on the plant and in the organza as well as on the amount of vegetal tissue ingested by the larvae. A. argillacea shows feeding preference for non-Bt cotton plants, in comparison to that on the Bt. However, factors such as temperature and exposure time may affect detection capacity and plant abandonment by the larvae and it results in lower ingestion of vegetal tissue. Such results are relevant to handle the resistance of Bt cotton cultivars to A. argillacea and they also enable determining how the cotton seeds mix will be a feasible handling option to hold back resistance evolution in A. argillacea populations on Bt cotton, when it is compared to other refuge strategies. The results can also be useful to determine which refuge distribution of plants is more effective for handling Bt cotton resistance to A. argillacea.
Assuntos
Animais , Temperatura , Gossypium/parasitologia , Comportamento Alimentar/fisiologia , Lepidópteros/fisiologia , Bioensaio , Controle Biológico de Vetores , Plantas Geneticamente Modificadas , Gossypium/genética , Lepidópteros/classificaçãoRESUMO
In Brazil, Spodoptera frugiperda (J. E. Smith) and Chrysodeixis includens (Walker) are important cotton pests and target of control of Bollgard II (Cry1Ac/Cry2Ab2) and WideStrike (Cry1Ac/Cry1F) cotton technologies. To subsidize an insect resistance management program, we conducted laboratory studies to evaluate the toxicity of these Bt cotton plants throughout larval development of S. frugiperda and C. includens. In bioassays with leaf disc, the efficacy of both Bt cotton plants against neonates was >80% for S. frugiperda and 100% for C. includens. However, S. frugiperda larvae that survived on Bt cotton had >76% of growth inhibition and stunting. In bioassays with S. frugiperda and C. includens larvae fed on non-Bt near-isoline during different time period (from 3 to 18 d) and then transferred to Bollgard II or WideStrike leaves showed that larval susceptibility decreased as larval age increased. For Bollgard II cotton, in all S. frugiperda instars, there were larvae that reached the pupal and adult stages. In contrast, on WideStrike cotton, a few larvae in fifth and sixth instar completed the biological cycle. For C. includens, some larvae in sixth instar originated adults in both Bt cotton plants. In conclusion, Bollgard II and WideStrike cotton technologies showed high efficacy against neonates of S. frugiperda and C. includens. However, the mortality of these species decreases as larval age increase, allowing insect survival in a possible seed mixture environment and favoring the resistance evolution.
Assuntos
Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Mariposas/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/genética , Brasil , Gossypium/metabolismo , Controle de Insetos/métodos , Larva/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , SpodopteraRESUMO
In 2009, 4088 hectares of genetically modified (GM) cotton were planted in Tolima (Colombia), however there is some uncertainty about containment measures needed to prevent the flow of pollen and seed from regulated GM fields into adjacent fields. In this study, the gene flow from GM cotton varieties to conventional or feral cotton plants via seed and pollen was evaluated. ImmunostripTM, PCR and ELISA assays were used to detect gene flow. Fifty six refuges, 27 fields with conventional cotton and four feral individuals of the enterprise "Remolinos Inc." located in El Espinal (Tolima) were analyzed in the first half of 2010. The results indicated seed mediated gene flow in 45 refuges (80.4 %) and 26 fields with conventional cotton (96 %), besides pollen mediated gene flow in one field with conventional cotton and nine refuges. All fields cultivated with conventional cotton showed gene flow from GM cotton. Two refuges and two feral individuals did not reveal gene flow from GM cotton.
En el 2009 se plantaron 4088 hectáreas de algodón genéticamente modificado en el departamento de Tolima (Colombia), sin embargo, hay ciertas incertidumbres acerca de las medidas de contención necesarias para impedir el movimiento de polen y semillas desde los campos GM regulados, hacia los campos adyacentes de cultivos convencionales. En este estudio se evaluó el flujo de genes mediado por polen y semillas desde variedades GM hacia variedades convencionales o individuos ferales, en el cultivo del algodón. Para detectar el flujo de genes se utilizaron ImmunostripTM, PCR y ELISA. Cincuenta y seis refugios, 27 campos con algodón convencional y cuatro individuos ferales de la empresa "Remolinos S.A." localizada en El Espinal (Tolima) fueron analizados en el primer semestre de 2010. Los resultados indicaron presencia de plantas GM en 45 refugios (80,4 %) y 26 campos de algodón convencional (96 %), además de un flujo génico mediado por polen en un campo de algodón convencional y nueve refugios. En todos los campos cultivados con algodón convencional se evidenció flujo de genes desde algodón GM. Solo en dos refugios y en dos individuos ferales no se evidenció flujo de genes desde algodón GM.
RESUMO
The two spotted spider mite, Tetranychus urticae Koch, is a nontarget herbivore of Bt-cotton, but acquires and accumulates higher levels of Cry toxin than that expressed by transgenic plants. This work investigated the development and reproduction of T. urticae and of the predator Phytoseiulus macropilis Banks, during three successive generations looking for potential nontarget effect. In addition, behavioral studies on feeding preference, oviposition, and predation were carried out on Bt and non-Bt cottons. The development and reproduction of T. urticae and P. macropilis was conducted using leaf discs of Bt and non-Bt cottons. Arena containing leaf discs from both cotton types connected by a slide coverslip were also used in the behavioral studies. Averages of the three generations showed that the Bt-cotton does not affect the development, survival of immature stages, and reproductive output of T. urticae and of the predator P. macropilis. Furthermore, the preference for feeding and oviposition of T. urticae and P. macropilis were similar on both cotton types. In addition, P. macropilis exhibited similar predatory behavior on T. urticae fed on both cotton types. Levels of Cry1Ac toxin in T. urticae was 3.97 times greater than that found in the Bt-cotton plants as determined using a ELISA test. Despite of the amount of toxin acquired by the prey (T. urticae), no detectable levels of Cry1Ac were found in the predatory mite P. macropilis.