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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 was 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 examined 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?

Summary

No differences in microbial maize litter breakdown were found between Bt maize and the isogenic variety in the three years of the trial. The Bt maize litter was not broken down any more slowly. However, some varietal differences were found.

The majority of the bacteria and fungi in the maize litter at the sampling times were metabolically active. The different maize varieties were not found to have any influence on the structure of the micro-organism communities. However, soil composition did have an effect.

Over the three years of the experiment, mycotoxin-producing fusaria were found to accumulate in the harvested material. Bt maize effects were observed in the second and third year. The significant differences were, however, restricted to specific times, so that there was no observable long-term impact on the density of mycotoxin-producing fusaria. Since the seeds had been treated with a range of different dressings, and some were therefore treated with different fungicides, this could have influenced the results.

Experiment description

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

Maize litter left on the field after harvesting

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

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

The litter was left on the trial field after threshing. 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 was collected from the trial field. DNA and RNA were isolated from these samples.

Characterisation of the composition of the bacterial and fungal communities

Genetic profiles (fingerprints) were produced using DNA from rotting plant material to investigate which bacterial and fungal groups accumulate during the breakdown of the maize litter. A gene was examined that is present in all bacteria/fungi, but which displays group-specific differences in terms of its DNA sequence. More detailed sequence comparisons were carried out for certain individual microbial groups.

To determine which bacteria/fungi are actively involved in breaking down the maize litter, genetic profiles (see above) were created using the ribosomal RNA of rotting plant material. Unlike the DNA examination, the RNA examination investigates the metabolically active micro-organisms whose DNA is strongly expressed and translated into proteins and which 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 ribosomal RNA genes. The results enable researchers to draw conclusions about the number of active bacteria of that group in the sample in question.

Measuring the density of mycotoxin-producing fusaria at harvest time and testing for potential Fusarium accumulation

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 genes. The results enable researchers to draw conclusions about the number of certain types of mycotoxin-producers in the sample in question.

Statistical analyses

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

Results

Soil respiration 2008: Amount of carbon dioxide released in 100 hours during litter breakdown by the micro-organisms.

Fingerprint pattern of the total and metabolically active bacterial community. The sample came from a transgenic maize plot four weeks after harvesting in 2008.

Fingerprint patterns of metabolically active bacterial communities in the different maize variants (sample taken 16/12/2008)

Density of mycotoxin-producing fusaria in the maize litter of the different maize variants following repeated cultivation

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. No significant differences were found between the varieties tested in 2010. No differences were found between the Bt maize and the isogenic variety in any of the three trial years.

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

The fingerprint patterns of the total bacterial flora (using DNA) largely corresponded to those of the metabolically active bacterial communities (using RNA). Most of the bacteria in the maize litter at the time of sampling were therefore 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. A clear correlation was found between the structure of the bacterial communities and the silt and sand fraction in the topsoil. A weaker correlation was also observed in relation to the pH level of the soil.

The fingerprint patterns of the entire and active communities were also the same for the fungal communities. This means that fungal microflora present on/in the litter when the samples were taken were evidently metabolically active. It was not possible to detect any link between the maize variety and the structure of the fungal communities for the entire trial period. As with the bacteria, soil quality was found to have an effect on the structure of the fungal communities in some cases, although this effect was not as strong as it was for the bacteria.

Measuring the density of mycotoxin-producing fusaria at harvest time and testing for potential Fusarium accumulation

The assessment quantified trichothecene- and fumonisin-producing fusaria. Mycotoxin-producing fusaria were found to accumulate in the harvested material over the course of the three-year experiment. Bt maize effects were observed in the second and third year. The significant differences were, however, restricted to specific times, so that there was no observable long-term impact on the density of mycotoxin-producing fusaria. Since the seeds had been treated with a range of different dressings, and some were therefore treated with different fungicides, this could have influenced the results.