Aug 6, 2004

Research Projects

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Structural analysis of fungal communities within the context of post-market monitoring

(2001 – 2004) Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Virology, Microbiology and Biosafety, Braunschweig

Topic

Soil micro-organisms are of great importance for soil quality and functioning material cycles in the agricultural ecosystem. In the context of post-market monitoring of genetically modified plants, microbial communities in the soil are to be studied in order to identify promptly any shifts.

In order to obtain a basis for the assessment of possible effects of transgenic plants on the community of soil micro-organisms, the project was to record the natural variability of fungal communities using the example of fungal communities in the root area of sugar beet.

The analysis was to cover in particular the extent of the effect of changing environmental conditions and agricultural techniques, so as to be able to identify at a later stage deviations that may have been caused by genetically modified plants.

Molecular methods for the characterisation of fungal communities were to be developed further so that they could be used in post-market monitoring.

It was shown that molecular biological methods for the analysis of microbial communities are better suited than traditional culture methods, since they require less effort and are more informative. Only an estimated one to ten per cent of bacteria living in the rhizosphere are identified using traditional culture techniques. The number of culturable fungi is not yet known.

Summary

The methods developed are suitable for demonstrating shifts in the structure of fungal communities from the soil and rhizosphere. The soil type was found to have a clear influence on the community structure of the fungi. The community structure was also found to be dependent on the plant. Seasonal shifts in the fungal communities in the soil were also found, depending on the sampling time/growth stage of the sugar beet.

It was not possible to demonstrate any influence of the variety on the soil fungal community. The strategies used are suitable for post-release monitoring, but not for post-market monitoring. Only the simultaneous cultivation of genetically modified plants, isogenic strains and conventional varieties on the same field can provide relevant data.

Experiment description

Between the plant world and the microbial community in the soil there is a strong reciprocal relationship. Through their roots, plants release a number of organic substances that produce stimulating and/or inhibiting effects on the micro-organisms in the root area. The impact of plants on the microbial communities is called the rhizosphere effect.

The composition of the substances released by the plant can vary between transgenic plants and non-transgenic plants. It is possible, for instance, for transgenic products to be deliberately released into the root area to fight microbial pathogens in the rhizosphere.

The studies took place in the root area of conventional sugar beet, since a release of herbicide-tolerant sugar beet could not proceed as planned. The variety test assessed the influence of variety and location on the species composition and abundance of the fungal community.

The focus was on the following working objectives:

Sampling and processing of soil samples

Developing practicable solutions for sampling and processing soil samples for fungal analysis using large sample volumes.

Optimisation of molecular genetic methods

Fingerprinting methods were to be tested and developed further for the characterisation of the fungal community in soil and rhizosphere samples. In particular, the aim was to develop suitable methods for series tests that would yield reproducible results.

Structure of the microbial community in the agro-ecosystem

The aim of the project was to supply answers to the following question: What is the “normal” structure of the microbial community in the agro-ecosystem, and outside which tolerance limits can a deviation be interpreted as a warning signal for possible negative effects of genetically modified plants on the fungal community? This presupposed that the natural fluctuation margin caused by environmental effects or agricultural measures would be determined in advance. Up to now there had been only a few studies to ascertain this “baseline”.

The present project is linked to the cross-section project on “Methods for the investigation of microbial communities for post-market monitoring of genetically modified plants” (2001-2006).

Results

Sampling and processing of soil samples

Mechanical pulping proved the most suitable method for breaking down fungal cell walls. A PCR method was also developed for processing DNA from difficult samples. With this method it was possible to duplicate large DNA fragments (semi-nested PCR). It was used to obtain fungal fingerprints (DGGE - denaturing gradient gel electrophoresis) from soil samples from 36 different long-term observation sites.

Established molecular methods for structural analysis of fungal communities were also used at several sites with different soil types and with five different varieties of sugar beet.

Optimisation of molecular genetic methods

Analysis of 18S-rDNA (a small sub-unit of fungal ribosomes) lent itself to determining the diversity of the fungal communities. These gene sequences are widespread in fungi. To assess the informativeness of the PCR DGGE fingerprinting method, a clone library and a collection of fungal isolates were set up. The electrophoretic mobility of the 18S fragments of fungal clones and isolates was compared with DGGE fingerprints of the fungal communities from the soil. It was possible to match most of the bands of clones and isolates to bands of DGGE fingerprints from the studied fungal communities.

Structure of the microbial community in the agro-ecosystem

During the 2002 vegetation period, four soil and two rhizosphere samplings were carried out. DNA was extracted from the soil/roots. Comparison of the DGGE patterns of the 18S rDNA fragments revealed a considerable impact of location on the composition of fungal communities in the soil.

Evaluation of the DGGE patterns revealed that the composition of the fungal community is heavily dependent on soil type.

It was not possible to demonstrate any influence of the sugar beet variety on the structure of fungal communities.