Novel methods for sampling and analysis of root exudates

Plant roots significantly influence the surrounding soil (i.e. rhizosphere) by exuding various organic compounds. This process called root exudation has a significant influence on soil chemistry, physics and biology and thus may largely determine the bioavailability of nutrients and toxic compounds. Previously, some research effort was put on the investigation of the root exudates, but in most cases only a small spectrum of the complete set of different compounds was investigated. Additionally, a majority of the exudates were obtained from hydroponic experiments, where the root exudate pattern may largely differ from a soil environment. The aim of this project is to implement high-end analytical techniques allowing the analysis of a large variety of target compound groups. Rhizobox systems will be implemented for the collection of exudates from soil-grown plants. This requires the adaptation of sampling devices in order to obtain unbiased sample concentrations at low volumes. Workpackage 1 will focus on: (i) the development of a protocol for extracting low molecular weight organic compounds (LMWOC) from rhizosphere soil; (ii) improvement of micro suction cups to minimize bias of the target compound concentration in the sample; (iii) improvement of a recently developed exudate collector system using alternative materials to avoid target compound adsorption. Workpackage 2 will focus on the implementation of high-end analytical approaches to analyse a broad spectrum of target compounds. This will include the: (i) implementation of miniaturized liquid chromatography systems with mass spectrometry detectors (LC-MS) for analysis of low sample volumes; (ii) design of online enrichment procedures; (iii) development, validation and application of a two-dimensional LC-MS system for simultaneous detection and quantification of the target compound groups. In Workpackage 3, a case study will be conduced to validate both the improved sampling methods as well as the application of the high-end analytical techniques. Additionally, the sampling techniques applied in the rhizobox system will be evaluated by comparison to aconventional hydroponic approach. The results of this project will be a major step to further understand the very complex processes in the rhizosphere of plants.

The sampling, detection and quantification of root exudates in the rhizosphere (i.e. the soil that is influenced by plant roots) is a challenging task due to very low sample volumes and partly also due to the very low concentrations of the target analytes. However, the elucidation of rhizosphere processes, that are to a very large extent depending on quantity and quality of root exudates, is extremely important for better understanding of plant nutrition, agricultural production and soil remediation. Regarding the sampling techniques a major progress has been achieved by the further development of the root exudates collector system. With this system it has been possible to sample exudates from roots of plants grown in a rhizobox system. In this specific rhizobox, the roots grow along a nylon membrane, that separates the roots from the soil solid phase, but allows the exchange of solutes. A membrane layer system has been put directly on the root layer and continuously rinsed with water or CaCl2 to collect the root exudates. To validate the system, the results were compared with the conventional hydroponic approach. In addition, micro suction cups were installed into the rhizosphere compartment of the rhizobox, which allowed the comparison of soil solution concentrations to the exudate release rates. Based on that, further insight into adsorption and/or degradation of root exudates can be derived. In parallel to the development of sampling approaches, analytical techniques for the detection and quantification of root exudates have been further developed. For the analysis of organic acids (e.g. citric acid), an LC-MS has been designed, which includes the conversion of the target compounds into a detectable form via derivatization. This approach was the basis for achieving extremely low detection limits (down to a few nMol), which are at least a factor 100 lower than previously used methods. For the analysis of amino acids, again an LC-MS method has been developed (hydrophilic interaction liquid chromatography combined with tandem mass spectrometry in multiple reaction monitoring (MRM) mode). With this method is was possible to achieve detection limits even in the sub- nMol range. For the detection of phytosiderophores, which are involved in the mobilisation of iron in soil, a capillary electrophoresis method has been established for the first time. With this approach is was possible to separate not only free phytosiderophores, but also their complexes with iron and other metals. The method has been applied to determine the stability constants of metal-phytosiderophore complexes. This major methodological achievement was the basis for the development of a recently started FWF-project, which targets on the clarification of rhizosphere processes of iron mobilisation by phytosiderophores.