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Breakdown of Bt toxin and effects on micro-organisms in the soil

(2005 – 2008) Federal Agricultural Research Centre (FAL) (since 2008 Johann Heinrich von Thünen Institute (vTI)), Institute of Agroecology; Braunschweig

Topic

The aim of this project is to examine the potential effects of the breakdown of diabrotica-resistant Bt maize on the soil. The main area of interest is the direct interaction between the plants and the soil bacteria that adhere to their roots. Molecular biological techniques will be used to compare the diversity of bacteria in the root area, the rhizosphere, of both Bt maize and conventional maize varieties.

The concentrations of Bt protein found in soil samples and plant remains after harvesting will also be quantified. This information will then serve as a database for other projects in this group, which are concerned with the potential effects on non-target organisms.

Summary

Quantifying the Cry3Bb1 protein

Soil analysis: Overall, the Cry3Bb1 levels in the soil are very low (less than one nanogram of toxin per gram of soil) compared with the levels in intact roots (100-370 µg/g). This indicates that the Cry3Bb1 protein is not released into the soil by the roots or is very quickly broken down in the soil.

Analysis of harvest residues: Four weeks after harvest, the concentrations of Cry3Bb1 measured in the roots that remained on the field were approximately one percent of the levels found in intact roots. Much lower concentrations were detected in the leaves left on the field.

Effects of the Cry3Bb1 protein on soil microbiology

Genetic fingerprints revealed no differences in the bacterial communities in the rhizosphere that could be attributed to the maize variety or the transgenic modification.

Experiment description

Quantification of the Cry3Bb1 protein

An ELISA test system was developed that can be used to detect small traces of the Cry3Bb1 protein from Diabrotica-resistant maize in environmental samples (detection limit 10 picograms Cry3Bb1 protein per gram of original material, dry weight). Using this method, soil samples and rotting plant remains (leaves and root remains from harvested plants) taken from the Bt maize field are tested for their Cry3Bb1 levels.

The soil samples are taken at four different growth stages of the maize (two early, one during flowering and one when ripe). In addition, four weeks after the harvest, soil samples and plant material (litter and root remains) are collected. The remaining harvest residues were tested again and analysed in subsequent years.

The soil samples are given to another group project for biotests with nematodes.

Effects of the Cry3Bb1 protein on soil microbiology

DNA is extracted from rhizosphere soil during the flowering period of the maize plants. A genetic fingerprinting method is used to compare the bacterial diversity in the rhizosphere of Bt maize with three conventional maize varieties.

Results

Quantification of the Cry3Bb1 protein

Soil analysis: In all three trial years Bt toxin was detected in the rhizosphere soil with an average concentration of 0.18 +/- 0.1 nanograms per gram, but was detected in only a few places in the bulk soil, as was to be expected. Over the course of the trial, Bt toxin was found increasingly frequently in the bulk soil, and in concentrations that were sometimes as high as 0.19 nanograms per gram. This was attributed to increasing numbers of roots in the soil on the trial area, as a result of the repeated maize cultivation (just 0.1 g of fine roots in one gram of soil could lead to a concentration of up to 30 ng toxin per gram of soil.)

Overall, the Cry3Bb1 levels in the soil are very low compared with the levels in intact roots, which are approx. 600,000 times higher. This indicates that Cry3Bb1 protein is broken down very quickly in the soil.

The Cry3Bb1 protein concentrations in the rhizosphere soil did not fluctuate significantly over the course of the vegetation period, with one exception in 2005. Significantly lower values were measured four weeks after harvest (on average 0.1 +/- 0.07 nanograms per gram of rhizosphere soil). This ties in with the expectation that the Cry3Bb1 protein is unstable and quickly breaks down in the plant remains left on the field after harvest.

Cry3Bb1 levels in rotting plant remains four weeks after harvest: root remains from the soil and root remains and leaves from the field surface. Concentration figures are based on the dry weight of the plant material.

Analysis of harvest residues: The analysis of harvest residues of leaves and root pieces four weeks after harvest found the highest Cry3Bb1 concentrations in the root remains left on the field (an average of 2.4 +/- 1.2 micrograms per gram of root). These concentrations are equivalent to around one percent of the levels found in intact roots. The lowest concentrations (average 0.2 micrograms per gram) were found in the leaves left on the field.

Six months after harvest the Cry3Bb1 concentrations in the roots left on the field had fallen to 0.3 and 18 nanograms per gram on average (in May 2006 and May 2007 respectively).

Unlike in previous years, in 2007 the whole trial field was ploughed up immediately after harvest so that four weeks after harvest it was only possible to measure values for the root remains in the soil. The Cry3Bb1 concentrations in the ploughed-in root remains were more than twice as high as the previous year (4 µg per g of root on average).

Persistence of the Cry3Bb1 protein in rotting root remains taken from the field surface following cultivation in 2006. Concentration figures are based on the dry weight. *below the detection limit

Effects of the Cry3Bb1 protein on soil microbiology

The genetic fingerprints did not reveal any differences in the bacterial communities in the rhizosphere that could be attributed to the maize variety or to the transgenic modification.

Neither did a more specific investigation of individual bacterial groups e.g. subgroups of proteobacteria and pseudomonads, find any differences.

The analysis results were very similar in all three years. This stability is attributed to the consistency of the factors plant type, soil type and plant age, which play a key role in determining the composition of the microbial community.