Sep 17, 2010

Research Projects

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Effects of Bt maize on micro-organisms that break down maize litter

(2008 – 2011) Leibniz Centre for Agricultural Landscape Research (ZALF), Institute of Landscape Matter Dynamics, Eberswalder Str. 84, 15374 Müncheberg

Topic

The aim of this project is to investigate the effects of the genetically modified Bt maize cultivar MON89034xMON88017, which produces three different Bt proteins, on bacteria and fungi that break down maize litter. Previous studies with single-resistance Bt maize have shown only minimal effects that are within the range of natural variation. Interactions resulting from the combination of different Bt genes could, however, increase the impact on micro-organisms and their activity.

Ploughless cultivation of Bt maize could lead to the maize litter being broken down more slowly and to an accumulation of cereal mould fungi (fusarium), which produce substances that are toxic to humans (mycotoxins).

This project will examine the following questions:

• Does the composition of the micro-organism community in the litter change when Bt maize is cultivated?
• Is there a change in the composition of the microflora that is actively involved in breaking down the maize litter?
• Do fusarium fungi that produce poisonous mycotoxins accumulate in the maize litter and is this more pronounced in crop rotations involving Bt maize than in those with conventional maize?

Experiment description

The genetically modified variety, its isogenic parent variety and two further conventional control varieties will be grown in a field trial. This method enables researchers to distinguish between potential Bt effects and varietal effects.

Sampling, measuring microbial maize litter breakdown and extraction of DNA and RNA

After the harvest, maize litter is removed from all the trial variants. It is dried and ground. To quantify the breakdown of the maize litter, the release of CO2 in a defined soil-ground litter mix is measured. DNA is extracted from the dried maize litter.

The litter is left on the trial field and some of it is ploughed into the topsoil during tillage. Four and eight weeks after harvesting and in the following spring rotting plant material with soil attached is collected from the trial field. DNA and RNA are isolated from these samples.

Characterisation of the composition of the bacterial and fungal communities

Genetic profiles are produced using DNA from rotting plant material to investigate which bacterial and fungal groups accumulate during the breakdown of the maize litter. Individual genes are examined that are present in all bacteria/fungi, but which are known to display group-specific differences in terms of their DNA sequence. More detailed sequence comparisons are carried out for certain individual microbial groups.

In order to discover whether various mycotoxin-producing fungi accumulate in the maize litter, a method is used that measures the number of copies of specific DNA sequences. The results enable researchers to draw conclusions about the number of those mycotoxin-producers in the sample in question.

Characterisation of the sections of the bacterial and fungal communities involved in the breakdown of maize litter

To determine which bacteria/fungi are actively involved in breaking down the maize litter, genetic profiles (see above) are created using the RNA of rotting plant material. Unlike the DNA examination, the RNA examination investigates the micro-organisms that play an active part in the metabolism process whose DNA is strongly expressed and translated into proteins and therefore play an important role in the litter breakdown.

To quantify the proportion of individual groups of micro-organisms involved in breaking down the maize litter, a method is used that measures the number of copies of specific RNA sequences. The results enable researchers to draw conclusions about the number of bacteria of that group in the sample in question.

Statistical analyses

A number of different statistical methods are used to correlate the data to the microbial communities and to those sections involved in the metabolic processes, and to the individual maize variants.

Results

Measuring microbial maize litter breakdown

The results from 2008 and 2009 showed that the variety of maize had a significant impact on litter breakdown. In 2008, CO2 release (soil respiration) was much higher for the transgenic Bt maize and isogenic parent variety than for the two other conventional varieties. This effect was less marked in 2009, but there was a significant difference between two conventional varieties. However, no differences were found between Bt maize and the isogenic variety.

Analysing the structure of the bacterial and fungal communities involved in the breakdown of maize litter

An analysis of the samples taken four weeks after the 2008 harvest and in March and April 2009 revealed that the fingerprint patterns of the total bacterial flora (using DNA) largely correspond to those of the metabolically active bacterial communities (using RNA). The majority of the bacteria in the maize litter at the time of sampling were evidently metabolically active.

The different maize varieties were not found to have any influence on the structure of the various micro-organism communities. However, soil composition did have an effect.

The maize straw from 2008 revealed only a small infestation with mycotoxin-forming fusaria. The assessment quantified trichothecene- and fumonisin-producing fusaria. No fumonisin-producing fusaria were detected in 2008. A comparison of the maize variants found no significant differences in numbers of trichothecene-producing fusaria. Four weeks after harvest, infestation by these fusaria fell by around a factor of ten.

The infestation level was higher in 2009. There was a significant difference between the two conventional varieties in terms of numbers of trichothecene-producing fusaria. There was a slightly significant difference in the number of fumonisin-producing fusaria found in the Bt maize and in the isogenic variety. However, only a small number of fumonisin genes were detected and only in a few isolated samples of the decomposing maize litter.