Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants

The aim of this study was to examine whether the terminal restriction fragm ent length polymorphism (T-RFLP) analysis represents an appropriate techniq ue for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RF LP method, a recently described fingerprinting technique, is based on termi nal restriction fragment length polymorphisms between distinct small-subuni t rRNA gene sequence types. This technique permits an automated quantificat ion of the fluorescence signal intensities of the individual terminal restr iction fragments (T-RFs) in a given community fingerprint pattern. The indi genous bacterial communities of three soil plots located within an agricult ural field of 110 m(2) were compared. The first site was planted with non-t ransgenic potato plants, while the other two were planted with transgenic G US and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundanc e values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatmen ts clearly revealed spatial and temporal effects, as well as space x time i nteraction effects, on the structural composition of the bacterial communit ies. T-RFs which showed the highest correlations to the discriminant factor s were not those T-RFs which showed the largest single variations between t he seven-sample means of individual plots. In summary, the T-RFLP technique , although a polymerase chain reaction-based method, proved to be a suitabl e technique for monitoring highly diverse soil microbial communities for ch anges over space and/or time. (C) 2000 Published by Elsevier Science B.V. o n behalf of the Federation of European Microbiological Societies.