Mar 20, 2009

Research Projects

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Movement of Bt toxin (Cry3Bb1) in the soil

(2005 – 2008) University of Göttingen, Institute of Applied Biotechnology in the Tropics (IBT e.V.)

Topic

The aim of this project is to examine how the deliberate release of genetically modified Bt maize varieties (Cry3Bb1) affects the soil in the release area. The precursor project has already shown that there is a close correlation between the soil surface characteristics and the binding of the Bt toxin (Cry1Ab) in the soil.

Therefore these projects will

• examine the physical and chemical properties of the soil on the site
• and determine the binding behaviour and movement of the Bt toxin in relation to the soil characteristics

Summary

The soils on the release site were very heterogeneous. Nevertheless, it was possible to make the following statements about the significance of soil type:

Soil characterisation

The nitrogen content of the soil on the Bt maize plots was no different from that of the soil on the conventional and isogenic plots. There was no significant variation in the level of organic material in the clay fraction of the topsoil within the trial field. The variations in cation exchange capacity (CEC) within the release site were attributed to the chemical and mineral composition of the clay fraction.

Binding of Cry3Bb1

Cry3Bb1 binds more strongly than Cry1Ab to the clay fractions of the plots under investigation. The Cry3Bb1 protein was found to bind more strongly to the clay fraction in the topsoil than in the subsoil. Furthermore, the subsoil showed greater affinity to Cry3Bb1 as the level of manganese oxides increased. Because of the heterogeneity of the soil, all samples from the clay fraction showed very different binding behaviour with regard to Cry3Bb1.

Determining the movement of the Cry3Bb1 toxin in the soil

Because of the results from the movement experiments with Cry3Bb1 in a concentration range relevant for the field, it is expected that Cry3Bb1 will move in soils with a low binding capacity. However, the type and extent of movement depend on the stability of the Cry proteins, the properties of the soil and the microbial activity in the soil in question.

Experiment description

Characterisation of the site

Soil samples were taken from the group’s release site at depths of 0-20 centimetres (top soil) and 40-60 centimetres (subsoil) as part of the soil mapping process for all 32 plots. The clay fraction (< 2 µm) and fine earth (< 2 mm) are extracted from these soil samples and are then characterised using soil analysis techniques and their binding behaviour is studied.

In addition, soil profiles were taken at three prominent locations in the release area (see diagram). Since the maize had already been sown at the time of sampling, the profiles were located outside the plots.

The soil is characterised by studying the following properties:

• Soil characteristics such as pH value, C/N ratio, carbonate content, particle size distribution and pore size distribution. The clay, silt and sand contents were determined as part of the particle size analysis. The cation exchange capacity (CEC, the capacity of a soil to exchange positively charged particles) of the samples was also determined.
• The chemico-mineralogical composition of the clay fraction
• The surface characteristics of the soil particles, e.g. surface charge and specific surface.

Binding of Cry3Bb1 and reversibility of the binding

To determine to what extent Cry3Bb1 binds to different soil fractions, 10 milligrams of clay and 50 milligrams of fine earth are mixed with 0-80 nanograms of Cry3Bb1 toxin. The Cry3Bb1 concentrations of the non-bound Bt toxin are measured by ELISA after 30 minutes. The investigations are conducted under sterile conditions to eliminate the possibility of microbiological breakdown of the Cry3Bb1 during the binding studies.

A further aim is to investigate the extent to which the temperature, pH value and different cations (sodium, calcium, magnesium) affect the binding of the Cry3Bb1 protein to the soil fractions.

Further experiments aim to clarify the conditions under which Cry3Bb1 is released from the soil.

In addition, the team studied the binding behaviour of Cry3Bb1 and Cry1Ab.

Determining the movement of the Cry3Bb1 toxin in the soil

In order to characterise the movement of Cry3Bb1 protein in the soil of the release site in greater detail, a column was filled with fine earth from the subsoil of the soil profile and the behaviour of the Cry3Bb1 protein in the soil was monitored.

Results

Characterisation of the site

Nitrogen content: In order to be able to detect any effect that Bt maize cultivation over several years might have on litter mineralization, the level of plant-available forms of nitrogen (N_min) on the release site was measured. The N_min values of the soil on the Bt maize plots are no different from those of the soil on the conventional and isogenic plots. In 2007 all the values were lower, because the maize plants were used to produce maize silage, so less crop residue entered the soil.

Particle size distribution: The topsoil (0-20 cm) had a clay content of 8-10 per cent and there was little variation within the trial field. The clay content in the subsoil was slightly higher than in the topsoil. The clay content in the subsoil in some plots was very high (30-40 %). The sand content was generally higher than the silt content in all plots.

The level of organic material in the clay fraction of the topsoil was in a typical farmland range of 2.1 to 3.3 per cent and there was no significant variation within the topsoil of the trial field.

The cation exchange capacity (CEC) of the fine earth in the topsoil varied slightly within the trial field. The subsoil of plots with a high clay content had a higher CEC and greater variation.

Unlike the topsoil, the subsoil, where there is less organic material, showed a strong correlation between clay content and CEC. The CEC of the clay fraction of the topsoil and subsoil was much higher than in the fine earth. The high CEC variation within the release site was attributed to the chemical and mineral composition of the clay fraction.

At between 5.0 and 6.0, the pH values of the soil were typical for a field. However, the topsoil had a lower pH than the subsoil, which can be attributed to top-down acidification.

Specific surface and surface charge : The specific surface area and negative surface charge of the topsoil are affected by both the clay content and the level of organic material. Because of homogenisation as a result of ploughing, no significant differences were found within the rows or plots. In the subsoil a clear correlation was observed between the clay content of the fine earth and both the surface area and the negative surface charge. This means that both surface values vary more in the subsoil because of different clay content levels. Because of their particle size, the components of the clay fraction demonstrated a greater specific surface than those of the fraction < 2 mm.

The chemical and mineral assessment measured the level of iron and manganese oxides and hydroxides. The iron oxide proportions can be used to draw conclusions about the age of the soil and crystallisation conditions.

Binding of Cry3Bb1 and reversibility of the binding

It was shown that the amount of bound Cry3B1 protein increases linearly when the Cry3Bb1 protein concentration in the solution which is added to the clay is increased (Diagram 1). Cry3Bb1 binds more strongly than Cry1Ab to the clay fractions of the plots under investigation (Diagram 2). It is not yet clear why the Cry proteins behave differently. It is, however, possible that the structural, chemical composition of the Cry proteins has a significant effect on the binding behaviour of the Cry proteins, as well as the characteristics of the soil constituents.

The Cry3Bb1 protein was found to bind more strongly to the clay fraction in the topsoil than in the subsoil. Furthermore, the subsoil showed greater affinity to Cry3Bb1 as the level of manganese oxides increased. Within the rows, only slight differences were observed in the binding behaviour of the clay fraction of the topsoil. The sample from one plot showed a much lower affinity to Cry3B1b.

Effect of soil science data on binding

Because of the differences in the range of variation between the topsoil and the subsoil, it is assumed that an unknown variable that is different in the topsoil and the subsoil has a significant influence on the other variables.

Determining the movement of the Cry3Bb1 toxin in the soil

In column experiments using fine earth, Cry3Bb1 was detected in the solution that passed through. So it must be assumed that movement of Cry3Bb1 is possible in soils with a low binding capacity. However, the type and extent of movement are highly dependent on the stability of the Cry proteins, the properties of the soil and the microbial activity in the soil.

The movement experiments with Cry3Bb1 were conducted with concentrations that are found in the field. Cry3Bb1 was detected in the solution that passed through. Beyond approx. 1.5 pore volume, the Cry3Bb1 concentration decreased. This may be because of a microbial breakdown of the Cry3Bb1 protein in the column.