Aug 2, 2005

Research Projects

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Breakdown of Bt maize in soils and impacts on micro-organisms

(2001 – 2004) Federal Agricultural Research Centre (FAL), Institute of Agroecology; Braunschweig

Topic

Proteins and DNA are normally decomposed very efficiently in the soil by micro-organisms. Therefore, on areas cultivated with Bt maize, the Bt toxin and the gene responsible for it (cry1ab) are probably also broken down by this group of organisms. On the other hand, the Bt toxin could itself have negative impacts on such organisms, or the recombinant genes could be transferred from the Bt plants to e.g. bacteria.

Within this project, research was carried out into these soil ecology questions with the aim of answering the following questions:

• It is possible that when Bt maize is cultivated outdoors, the Bt toxin enters the soil of the root zone (rhizosphere) via the roots or that when the plant material rots, the toxin enters the soil that adheres to these plant remains (residue sphere). Are the micro-organisms found in the rhizosphere of Bt maize (Mon810) different from those found in the rhizosphere of non-GM varieties?
• Can such Bt toxin releases in the soil be detected, and if so, how much Bt toxin can enter the soil by this means and how long does it remain there?
• Does the Bt gene (cry 1Ab) remain in the soils of crop areas beyond the vegetation period, thereby leading to an unnatural accumulation of this genetic material?

Summary

The expression of the Bt toxin in plant roots probably leads to minor structural changes within the community of rhizosphere bacteria. These changes are smaller than changes caused by different types of soil, plant age or variable field conditions.

When Bt maize is cultivated, the Bt toxin enters the soil; the main source is the root remains of the harvested maize plants. Small amounts of the Bt toxin remain in the soil for longer than one vegetation period and could lead to an accumulation of the Bt toxin in the soil in the case of monocultures.

The released Bt toxin quantities are below the response threshold for the target organisms – any effect on non-target organisms is therefore unlikely.

Transgenes from Mon810 can be detected in rotting plants after the harvest – a contribution to horizontal gene transfer is, however, very unlikely.

Experiment description

Sampling

Various samples were taken at two locations with Bt maize and an isogenic strain during the vegetation period, immediately after the harvest and at the end of the winter period. The samples were taken from the maize rhizosphere, from root remains and from rotting plant material, open soil and rhizosphere soil. The experiments covered three vegetation periods. The samples are from maize monoculture fields, since it is easier to demonstrate a possible build-up of the toxin in these fields.

Sample analyses

• Micro-organism communities. The DNA present in the soil samples was extracted and examined for diversity and any possible change in the composition of the soil micro-organism community. A particularly sensitive procedure was used (PCR-SSCP), which functions in a similar way to the genetic fingerprinting used in criminal investigations.
• Toxin content. The sieved soil samples were analysed for their Bt toxin content (ELISA).
• Detection of Bt genes. The DNA present in the soil samples was extracted and examined to see whether it is possible using PCR to detect the origin of Bt genes: from natural Bt producers (Bacillus thuringiensis) in farmland or from the Bt maize.

Results

Micro-organism communities. Using molecular biological methods (PCR-SSCP of 16S rRNA genes) it was possible to show the diversity of bacteria in the root area (rhizosphere) as well as in genetic fingerprints.

The genetic fingerprints of the rhizosphere bacteria revealed with decreasing intensity:

• A change depending on the age of the plant
• Differences between the same varieties at two different sites
• Differences in the same field, as a result of different growth conditions (field heterogeneity) and
• A change characteristic of Mon810

Toxin content. Using a procedure developed for detecting Bt in soil (ELISA) it was possible to detect even small traces of Bt or its intermediate degradation products (70 pg g-1) in the soil. In 2002 for the first time, soil samples from several plots were tested using this method. The samples were taken four times per vegetation period, always at precisely defined maize development stages. As expected, the measurements in the soil attached to the roots were usually noticeably higher than those taken from open soil. There were also differences depending on the age of the plant.

In the second cultivation year, all measured Bt values were significantly higher than those of 2002 at both sites. The increase in toxin content was five or seven times the previous year’s level, depending on the site. Even in soil samples taken in April 2003, i.e. before the next planting season, something could still be detected. Although the measured values in the soil increased from one year to the next, they are still very low and, by way of comparison, are equivalent to just one thousandth of the Bt concentrations typically measured in the roots of Bt maize.

The project also studied rotting plant remains (leaves and roots), which had stayed on the field for about seven months after the harvest. The values in these plant remains were about ten per cent of those found in the roots of intact Bt plants. The results also demonstrated that the Bt toxin is evidently broken down more slowly in root remains than in rotting leaves. In further samples taken in the summer the measurements from the plant remains samples fell significantly.

Detection of Bt genes. A PCR method for detecting transgenic cry1Ab genes was established. Transgenes were still found in plant remains from the previous vegetation period.