The Combination of DNA Analytical Methods and Micropedology to Investigate Microorganisms in Undisturbed Soil Samples.
Thilo Eickhorst and Rolf Tippkötter. University of Bremen, Institute of Soil Science, Leobener Str. UFT, Bremen, 28359, Germany
Terrestrial ecosystems participate in different ways in the global metabolic cycle, in which most of the decomposition of substances is done by soil microorganisms. Due to the typical spatial mobility and distinctive life cycles of soil-inhabiting microorganisms, the detection and identification of those being important to the microbiological processes must be as precise as possible. The soil structure is formed by a complex composition of minerals and organic matter which leads to a wide range of living conditions. Therefore the soil matrix contains a lot of different microhabitats for soil microbiota. The present state of knowledge regarding the biodiversity of soil bacteria and the spatial distribution of bacterial cells in the soil matrix is as negligible as the understanding of the interaction between the soil structure and microorganisms. Present DNA analytical methods such as Fluorescence In Situ Hybridization (FISH) have therefore been introduced to improve the characterization of microbial biocoenoses without the need for conventional cultivation methods. Therefore a method for in situ detection of microorganisms in a soil matrix in two- and three dimensional arrangement combining molecular and micropedological techniques was developed. The combination of FISH and embedding soil structure with resin is a new method in soil molecular biology which allows the localisation and identification of the dynamics of microorganisms in the undisturbed soil matrix. After a series of pre-experiments on different soil suspensions FISH is used for structured soil samples in small sample rings. Using fluorescent labelled 16S rRNA targeted oligonucleotide probes soil microorganisms are stained specifically and visualized in the soil matrix using fluorescence microscopy. Different oligonucleotide probes targeted to certain phylogenetic levels are applied to detected dominant populations. Therefore the “Top to Bottom”-approach will be followed by detecting microbes from domain to genus level. Cross-checking was done by dyeing with DAPI as well as the use of different excitation filters during fluorescence microscopy. Embedding structured soil samples in polyester resin enables the production of polished blocks, which allow a spatial assignment of bacteria marked by FISH. Two possibilities to use FISH in this approach are compared: hybridization of soil structure (a) before and (b) after impregnation with resin. Furthermore micropedological methods allow a detailed record of the microstructure of the soil samples investigated by this novel approach. Therefore thin sections and polished blocks are observed with techniques of light- and epifluorescence microscopy as well as digital image analysis. Within this contribution the application of the novel approach is shown exemplified for the investigation of microbial dynamics in paddy soils related to soil structure dynamics caused by seasonal flooding and desiccation cycles. Undisturbed paddy soil samples of three different textures from southeast China are used to investigate the distribution of bacteria and archaea in the soil matrix at different stages during rice growth. These analyses point out clear interactions of microstructure and microbial populations particularly with regard to the plough pan compared with the puddled top soil. For the first time FISH was used for spatial detection of microorganisms in undisturbed soil which enabled the visualization of new aspects of soil. This method can be used for specific localization and characterization of microorganisms and their dynamics in the soil matrix. Applying FISH especially in combination with micropedological analysis may lead to a better understanding of structure and function of microbial communities. By using various oligonucleotide probes this technique can be used for quantitative as well as qualitative analysis of the diversity of bacteria and archea in the soil matrix. Regarding to the influence of soil structure on soil microbes more information about their activity, their function and spatial distribution of functional groups in the soil matrix are expected, which is important for the analysis of interrelations between the various processes in soils.