Effects of transgenic T4 lysozyme potato plants on micro-organisms in the soil – focusing on bacteria and fungi in the rhizosphere

Topic

Phytopathogenic bacteria can cause extensive crop failure and damage to potato tubers during cultivation and storage.

A transgenic potato has been engineered, which shows a particular resistance to Erwinia carotovora, the pathogen which causes blackleg and soft rot.

Resistance was produced by transferring the gene for the enzyme lysozyme, which dissolves bacterial cell walls, from T4 bacteriophage into the plant genome.

As T4 lysozyme does not only affect phytopathogenic bacteria, the project aimed to investigate the potential impact on communities of bacteria and fungi in the rhizosphere . The T4 lysozyme produced in the transgenic potato plant is transported into the apoplast, the cell wall space and intercellular space, and can also be detected outside the roots. For this reason, in addition to the micro-organisms in the rhizosphere, the fungi growing in the intercellular space of the plant (endophytic) were also considered.

Summary

During the two-year field trials, bacterial and fungal communities in the rhizosphere showed a high degree of stability. No effects of T4 lysozyme were observed. Differences within the communities did occur, however, depending on the stage of development of the plants and the trial year.

No obvious effects were observed in the endophytic fungi. To detect the potential effects of the T4 lysozyme on endophytes, classic (fungi cultivation) and molecular methods (fingerprinting ) were applied using clone libraries.

Experiment description

Within the scope of the field trials a comparative analysis was conducted on soil from the immediate root area of various transgenic strains, one transgenic control strain without the T4 lysozyme gene, and the non-transgenic strain. The two-year field trials were conducted at Groß Lüsewitz, Rostock.

Analysis of the communities of micro-organisms in the rhizosphere

The structure of the community of micro-organisms in the rhizophere was analysed using genetic fingerpinting. In contrast to classic methods, the molecular genetic approach enables non-cultivable micro-organisms to be identified as well (classic method: bacteria must be grown on a culture medium before they can be identified).

Different DNA sampling techniques were examined prior to molecular biological identification of the fungal and bacterial communities. The DNA was extracted either directly from the soil particles attached to the roots or after first extracting the soil particles from the roots.

To investigate the bacterial communities, genes which code for the 16S rRNA (a small subunit of the bacterial ribosomes ), were then amplified using specific primers . By using other bacterial group-specific primers it was also possible to record bacterial groups such as Actinomycetales, alpha- and beta-Proteobacteria and pseudomonads, whose DNA can amount to less than one percent of the total DNA. The fragments obtained were then separated electrophoretically in a gradient gel (DGGE). The resulting fingerprints for each sample could then be analysed and compared.

To investigate the fungal communities, genes which code for the 18S rRNA (small subunit of the fungal ribosomes) were amplified using specific primers.

Investigating the endophytic fungi

The endophytic fungi were isolated on the one hand using classic methods after sterilisation of the root surfaces (to kill off the micro-organisms and destroy the DNA on the root surface) and subsequent cultivation of the root segments.
In addition, a culture-independent method was used to extract the DNA from the roots, the surface of which had been sterilised, amplify it using a fungi-specific primer and then conduct molecular genetic analysis.

Results

Bacterial community

It was shown that the bacterial communities of the rhizosphere have a high degree of stability. Differences were generally determined by the season and year. There was no evidence of strain-determined differences, i.e. differences between different transgenic strains and the non-transgenic parent variety Désirée. The investigations with bacterial group-specific primers for alpha- and beta-Proteobacteria and for pseudomonads and Actinobacteria also showed no evidence of strain-related differences.

Fungal communities

In contrast to the bacterial communities in the rhizosphere, a somewhat higher degree of variation was found in the fungal communities across the trial variants. This can presumably be attributed to the distribution of the fungi in the habitat. Seasonal variations were also found for the fungal communities. Strain-related differences were not detectable.

Endophytic fungi

Root colonisation by endophytic fungi was investigated comparatively using classic and molecular techniques for the first time. However, only one sampling date in 2000 was considered and the non-transgenic control Désirée was compared with one transgenic highly lysozyme-producing strain. A high density of colonisation was found for both potato strains. Using the classic isolation method, significant differences in the colonization of the two strains with the fungi Verticillium dahliae and Colletotrichum destructans were detected for the first time. The latter were also detected in the fingerprints of the roots. The higher colonisation by Verticillium dahliae determined using classic methods could not, however, be verified by the molecular biological methods.

Overall it was shown that assured results on the effects of new plant strains on bacterial and fungal communities in the soil require not only studies lasting several years but also a combination of classic and molecular biological methods of investigation.