Oct 30, 2009
Effect of zeaxanthin potatoes on soil life – bacterial and fungal diversity in the root zone
(2005 – 2008) Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biosafety; since 2008 Julius Kühn Institute (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig
The aim of the project was to examine the unintentional effects of cultivating transgenic potatoes with modified carotenoid composition (zeaxanthin-enriched) on the bacterial and fungal community in the root area. Of particular interest was whether other metabolic processes and potato root exudates change as a result of the genetic modification and whether this affects the structures and functional diversity of the fungal and bacterial communities.
There was also the question of whether there is an impact on the abundance and diversity of beneficial micro-organisms (in this case antagonists of harmful bacteria and fungi).
A combination of established approaches and new monitoring methods were used for the analysis.
- It was shown in this project that the transgenic plants had effects on both the bacterial and fungal communities in the root area and on the tuber surface. In all cases, however, the varietal effect of the different potato varieties was greater than the effect of the genetic modification.
- Environmental factors such as location, year and plant development stage had the greatest effect on the composition of the bacterial communities and their antagonistic potential.
- For the transgenic lines, the number of bacterial antagonists was always within the range of natural varietal variability.
- The composition of the fungal community in the root area and inside the root was largely influenced by location.
Soil samples from release trials
The release trials were being conducted at trial sites in Roggenstein (Bavaria; 2005, 2007) and Oberviehhausen (Bavaria; 2006, 2007). Samples were taken from the two transgenic lines (sense and antisense), one non-transgenic parent variety and four further conventional varieties at different stages of development of the plant (young, flowering and dying).
Samples for both the bacterial and fungal investigations were taken from the root area (rhizosphere), directly from the tuber surface (geocaulosphere) and from the internal root tissue (endorhiza). The micro-organism community was analysed using traditional culture techniques and culture-independent molecular studies of directly extracted DNA.
Culture-dependent studies: The culture-dependant method recorded only those bacteria that can grow on and colonise culture media. Fungus colonies were also isolated. This was the method used to identify colony-forming units.
Antagonistic activity: Once the number of colonies has been determined, the dominant bacterial colonies are isolated and tested for their antagonistic effect on three of the potato’s key pathogens (Rhizoctonia, Verticillium and Phytophthora).
Among the fungi, Trichoderma, an important indicator group, were of particular interest. Trichoderma frequently show antagonistic activity towards plant pathogens. They can inhibit fungal growth by producing countless antibiotics or by hyperparasitism.
Culture-independent studies. These methods record the entire bacterial and fungal community. Characteristic segments of ribosomal RNA genes (16S rRNA gene fragments in bacteria and the ITS1 region in fungi) were amplified using PCR techniques and then separated using electrophoretic procedures. The separated DNA segments were marked to make them visible, which reveals a typical banding pattern (a genetic fingerprint).
Structural diversity: By comparing these fingerprints, statements can be made about the structural diversity of the bacterial and fungal community, the variability between repetitions, the differences between transgenic strains and the parent variety and between varieties.
Functional diversity: PCR and Southern Blot hybridisation were also used to identify the genes in the extracted DNA that code for proteins for the production of antibiotics.
The first field trial took place in 2005 in Roggenstein. The trial plants were planted in randomly distributed plots, with six repetitions per variety. Deliberate release approval was received too late for this site in 2005, but the conventional varieties were still planted so that the sites could be compared. The Oberviehhausen site was added in 2006. The experimental approach was the same as that used the previous year in Roggenstein.
The trial site in Roggenstein
To record the micro-organism community in the root area (rhizosphere) of the potato plant, the roots are cut off and then “washed”.
The outer skin of the tubers is removed by hand.
Here four different bacteria are being tested for their antagonistic potential against the potato pathogen Phytophthora infestans. The bacterial isolates are placed on an agar plate in lines and P.infestans is placed in between. The size of the zone of inhibition developing around the bacteria is a measurement of their antagonistic potential against the pathogen.
Here four different fungal isolates are being tested for their antagonistic effect against the potato pathogen Verticillium dahliae, also a fungus.
Samples were taken from both sites at three stages of development. Root material from five plants per plot was combined to obtain a composite sample. In 2006, tuber samples were taken in addition to the roots. Five tubers of equivalent size per variety were combined to form a composite sample.
Investigating the bacterial community (Smalla, BBA Braunschweig)
- in the root area (rhizosphere): Significant differences were found during flowering (EC60) in Roggenstein (2005) and for young and dying plants (EC30 and EC90) in Oberviehhausen (2006). These differences occurred both between the transgenic lines and some conventional varieties, and between various conventional varieties.
- on the tuber surface (geocaulosphere): The surface of the tubers was examined at the Oberviehhausen (2006) and Roggenstein (2007) sites when the tubers were fully formed. At both sites, the colony-forming units were identified first. Significant differences were found at both sites both between individual conventional varieties and between the transgenic varieties and certain conventional varieties.
Antagonistic activity: Some significant differences in the total number of antagonists in the root area were found when viewed across all three sampling periods. These differences occurred both between the Baltica parent variety and the transgenic lines, and between various commercial varieties. At the Roggenstein site, these differences were due primarily to the high level of antagonists found in the Désirée variety. Overall, however, the number of antagonists for the transgenic lines was always within the range of natural varietal variability.
Culture-independent analysis: The bacteria with antagonistic activity from the root area underwent phenotypic and genotypic characterisation. The genotypic analysis found clear differences in the composition of antagonists as a function of the location. Whereas a large proportion of antagonists from the Roggenstein site belonged to the Pseudomonas genus (gammaproteobacteria; 47.2 %), the antagonists from the Oberviehhausen site belonged primarily to the Streptomyces (46.7 %) and Bacillus/Firmicutes (29.2%) genera. Location had the greatest effect on the level and diversity of antagonists in the rhizosphere. By contrast, the effect of the plant genetic modification was within the range of natural varietal variability for the trait under investigation.
Investigating the fungal community (Berg Group, TU Graz)
Culture-dependent studies in the root zone (rhizosphere): Average fungal concentrations of 4 x 105 per gram of fresh root mass were obtained at both sites in all three trial years, which confirms the stability of the colonisation. Slight variations between sampling dates were found, but these were not significant. The transgenic modification was not found to have an impact on the fungal flora.
- inside the root (endorhizosphere): Very low fungal concentrations of between 0 and 1 x 103 colony-forming units per gram of fresh root mass were found inside the root in all three years. The fungal colonisation of the endorhizosphere in the samples examined was extremely diverse. No correlation could be found between the transgenic modification and the colonisation inside the root.
- on the tuber surface (geocaulosphere): Samples were first taken from the tuber surface in the second year of the project in Oberviehhausen. No significant differences were found in the fungal colonisation of transgenic lines and conventional varieties.
Antagonistic activity: Fungal isolates with antagonistic in vitro activity against the three potato pathogens were detected in every sample examined. However, the level of antagonistic fungal isolates varied considerably at the two sites. It was not therefore possible to detect an effect for this parameter caused by the genetic modification vis-à-vis the parent variety.
Culture-independent analysis: The structural diversity analysis found that microfungi make excellent indicator organisms for evaluating impacts on the microbial community. A comparison of the commercial varieties with the transgenic varieties at different stages of development in different soils found that location has the greatest influence on the structural diversity of the fungal community.
Specific primers were developed for the fungal genus Trichoderma that can be used for quick and easy molecular biological detection in environmental samples. Major differences were found between the two sites. In Oberviehhausen the Trichoderma genus was very common and displayed a high level of diversity. By contrast, in Roggenstein, Trichoderma numbers were three times lower with a smaller number of species. The differences between the varieties were minor and not statistically significant. The same thing applied to the different developmental stages of the potatoes.
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Bundesministerium für Bildung und Forschung
Einfluss der Freisetzung von transgenen Kartoffeln auf die Funktionalität der Bodenmikroflora, und Evaluierung der Verbreitung von Samen
Untersuchungen an Bakterien:
PD Dr. Kornelia Smalla
Julius Kühn-Institut (JKI)
Institut für Epidemiologie und Pathogendiagnostik
Tel. 0531 299 3814
Untersuchungen an Pilzen:
O. Univ.-Prof. Dr. Gabriele Berg
Technische Universität Graz
Institut für Umweltbiotechnologie
Tel: +43 (0) 316 873-8819
Weinert N., Meincke R., Gottwald C., Heuer H., Gomes N. C. M., Schloter M., Berg G. und Smalla K. (2009) Rhizosphere Communities of Genetically Modified Zeaxanthin-Accumulating Potato Plants and Their Parent Cultivar Differ Less than Those of Different Potato Cultivars. Applied and Environmental Micobiology, June 2009, p. 3859-3865 Vol. 75, No. 12 doi:10.1128/AEM.00414-09
Weinert N., Meincke R., Gottwald C., Radl V., Dong X, Schloter M., Berg G. und Smalla K. (2009) Effects of genetically modified potatoes with increased zeaxanthin content on the abundance and diversity of rhizobacteria with in vitro antagonistic activity do not exceed natural variability among cultivars. Plant and Soil DOI 10.1007/s11104-009-0024-z