COMPARISON OF EAR DAMAGE CAUSED BY CATERPILLAR PESTS IN TRANSGENIC (BT) MAIZE HYBRIDS AND CONVENTIONAL MAIZE HYBRIDS

The use of transgenic (Bt) maize hybrids has been an important tool to minimize ear damages caused by the caterpillar pests Helicoverpa zea and Spodoptera frugiperda (Lepidoptera: Noctuidae). The objective of this work was to evaluate ear damages caused by caterpillar pests in maize hybrids with Bt technologies and in their respective conventional versions (non-Bt) for control of caterpillar pests that attack maize ears in the state of São Paulo, Brazil. Experiments were carried out in four regions of the state, in two summer crops (2009/10 and 2010/11), using a randomized block design with 6x2 factorial arrangements. The first factor was the hybrids and the second factor was the use or not of Bt technology. During harvesting, the percentage of damaged ears was evaluated and damage scores were attributed to a sample of 20 ears per plot. It was found that: (i) YieldGard and Total Liberty (both Cry 1Ab), Herculex (Cry 1F), VTPRO (Cry 1A.105 + Cry2Ab2) and Viptera (VIP3Aa20) technologies, regardless of the hybrid and the season crop, significantly reduce the ear damages and the percentage of damaged ears; (ii) hybrids expressing the Cry 1Ab protein are more damaged by caterpillar pests compared with other technologies; and (iii) there is great variability among hybrids when expressing toxins, even among those hybrids with the same Bt event.

Highly sensitive immunosensing platform for one-step detection of genetically modified crops

The wide cultivation of genetically modified (GM) insect-resistant crops has raised concerns on the risks to the eco-environment resulting from a release of Cry proteins. Therefore, it is vital to develop a method for the quantification of GM crops. Herein, A highly sensitive immunosensing platform has been developed for both colorimetric and chemiluminescent (CL) detection of Cry 1Ab using dual-functionalized gold nanoparticles (AuNPs) as signal amplification nanoprobes for the first time. In this work, anti-Cry 1Ab monoclonal antibody and horseradish peroxidase (HRP) are simultaneously functionalized on the surface of AuNPs with an exceptionally simple synthesis method. Combined with immunomagnetic separation, this immunosensing platform based on colorimetric method could detect Cry 1Ab in one step in a linear range from 1.0 to 40 ng mL−1 within 1.5 h, with a limit of detection of 0.50 ng mL−1. The sensitivity of fabricated nanoprobes was 15.3 times higher than that using commercial HRP-conjugated antibody. Meanwhile, the fabricated nanoprobes coupled with CL detection was successfully applied for Cry 1Ab detection with a minimum detection concentration of 0.050 ng mL−1 within a linear range of 0.10–20 ng mL−1. The proposed approach was validated with genuine GM crops, and the results showed a good correlation coefficient of 0.9906 compared to those of a commercial ELISA kit. Compared with ELISA, the developed immunosensing platform significantly simplified the assay procedure and shortened the analytical time, thus providing a new platform for the detection of genetically modified crops with high sensitivity, rapidity and simplicity.

Eficácia de milho transgênico tratado com inseticida no controle da lagarta-do-cartucho no milho safrinha no estado de São Paulo, Brasil

RESUMO Diversas tecnologias de milho geneticamente modificado (Bt) foram liberadas comercialmente desde 2007 visando principalmente o controle da lagarta-do-cartucho, Spodoptera frugiperda (Lepidoptera: Noctuidae). O objetivo deste trabalho foi avaliar a eficiência de híbridos transgênicos (Bt) e convencionais (não Bt) no controle lagarta-do-cartucho, submetidas ou não a inseticidas. Para isso, foram conduzidos ensaios em três localidades do estado de São Paulo nas safrinhas de 2009, 2010 e 2011, com delineamento experimental de blocos casualizados, em esquema fatorial 5x4, 5x4 e 8x4, respectivamente. O primeiro fator correspondeu ao número de híbridos comerciais de diferentes empresas. O segundo fator foi constituído pela utilização de diferentes manejos de controle do inseto. Para a avaliação dos danos ocasionados pela lagarta-do-cartucho, foram realizadas amostragens ao acaso de 20 plantas por parcela, aplicando-se uma escala de notas visuais, atribuindo notas que variam de 0 (sem dano) a 9 (cartucho totalmente destruído) e obtido a produtividade de grãos. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Tukey a 5% de probabilidade, para cada parâmetro avaliado em cada localidade. Os híbridos transgênicos proporcionaram redução nas notas de danos atribuídas a lagarta-do-cartucho e as proteínas Cry 1F, Cry 1A105 e VIP3Aa20 foram as mais eficientes na redução do dano foliar. A pulverização mostrou-se uma estratégia eficiente em reduzir os danos foliar provocados pela praga. A combinação da pulverização com híbrido transgênico, de modo geral, mostrou ser uma boa estratégia para redução de dano foliar, especialmente quando foi utilizada a proteína Cry 1Ab, comprovadamente de menor eficiência para redução dos danos causados pela praga.

Cry 1Ab and Cry 9C Transgenic Corn Hybrid Effects on Laboratory Populations of Indianmeal Moth (Lepidoptera: Pyralidae) and Angoumois Grain Moth (Lepidoptera: Gelechiidae)

Several Bacillus thuringiensis Berliner (Bt) Cry proteins including Cry 1Ab, Cry 1Ac, and Cry 9C, have been observed at relatively high levels in Bt corn grain using the CaMV 35S promoter. Thus, a laboratory experiment was conducted to quantify the impact of DeKalb 679 BTY Cry 1 Ab (MON 810) and Garst 8600 BLT Cry 9C (CBH 351) transgenic grain on Indianmeal moth, Plodia interpunctella (Hübner), and Angoumois grain moth, Sitotroga cerealella (Olivier), survival, development and fecundity. Eggs of Indianmeal moth or Angoumois grain moth were added to cracked or whole kernel corn. Emergence and fecundity were recorded for 5 wks. Emergence and fecundity of both moth species was reduced on both Cry 1Ab and Cry 9C-transformed corn, but only Cry 1 Ab-transformed corn delayed development of Indianmeal moth. Results indicate that populations of these moths may be negatively impacted in grain bins by Bt corn hybrids and that lepidopteran populations should be monitored in field-scale assays to examine the effects of the presence of Bt corn hybrids on insects in storage environments.

Study of the Basis for Toxicity and Specificity of Bacillus thuringiensis d-Endotoxins

The report contains three parts which summarizes the three years achievements of the three participating research groups; The Weizmann group, Tel-Aviv group and Purdue group. The firs part describes the achievements obtained by Shai's group toward the elucidation of the mechanism of membrane insertion and the structural organization of the pores formed by the Cry3A and Cry1Ac B. thuringiensis d-endotoxins. For that purpose Shai's group synthesized, fluorescently labeled and structurally and functionally characterized peptides corresponding to the seven helices that compose the pore-forming domain of Cry3A toxin, including mutants peptides and the hairpin a4G-a5 of both Cry3A and Cry 1Ac toxins composed of a4, a5 and the loop connecting a4-a5. Among the synthesized peptides were three mutated a4 helices based on site directed mutagenesis done at Aronson's group that decreased or increased Cry 1Ac toxicity. The results of these studies are consistent with a situation in which only helices a4 anda5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like ribs of an umbrella (the "umbrella model"). In order to test this model Shai's group synthesized the helical hairpin a4<-->a5 of both Cry3A and Cry 1 Ac toxins, as well. Initial functional and structural studies showed direct correlation between the properties of the mutated helices and the mutated Cry1Ac. Based on Shai's findings that a4 is the second helix besides a5 that insert into the membrane, Aronson and colleagues performed extensive mutation on this helix in the CrylAc toxin, as well as in the loop connecting helices 4 and 5, and helix 3 (part two of the report). In addition, Aronson performed studies on the effect of mutations or type of insect which influence the oligomerization either the Cry 1Ab or Cry 1Ac toxins with vesicles prepared from BBMV. In the third part of the report Zilberstein's and Sneh's groups describe their studies on the three domains of Cry 1C, Cry 1E and crylAc and their interaction with the epithelial membrane of the larval midgut. In these studies they cloned all three domains and combinations of two domains, as well as cloning of the pore forming domain alone and studying its interaction with BBMV. In addition they investigated binding of Cry1E toxin and Cry1E domains to BBMV prepared from resistant (R) or sensitive larvae. Finally they initiated expression of the loop a4G<-->a5 Cry3A in E. coli to be compared with the synthetic one done by Shai's group as a basis to develop a system to express all possible pairs for structural and functional studies by Shai's group (together with Y. Shai).