DNA-Based Stable Isotope Probing for Identifying Active Bacterial Endophytes in Potato

DNA-based stable isotope probing (SIP) was introduced as a powerful tool to achieve a deeper insight of microbial processes through identifying relevant organisms that determine nutrient cycling and metabolize plant-derived compounds. Since its advent, it has been mainly used for studying micro-organisms in terrestrial ecosystems including the rhizosphere, but the endosphere has not been considered so far. Here we present a baseline study in which we used a 13CO2 (99 atom-%, 350 ppm) pulse labeling incubation experiment to identify active bacterial endophytes in two Solanum tuberosum L. cultivars, Desirée and Merkur (Rasche et al., 2009). Active bacterial endophytes were determined by SIP along with 16S rRNA gene-based community analysis including terminal restriction fragment length polymorphism analysis and generation of gene libraries. Our results show that the 13C label shuttles from plant assimilates to the endophytic bacterial community. In the 13C-enriched SIP fractions, a specific terminal restriction fragment became abundant, which could be clearly affiliated with Acinetobacter and Acidovorax sp. in Merkur and Desirée, respectively, indicating cultivar-specific distinctions in the 13C label flow. In summary, the presented DNA-based SIP approach was clearly suited to determine active bacterial endophytes in potato shoots which led to the overall conclusion that DNA-based SIP studies possess a very high potential to advance our current understanding of the complex nature of plant–microbe interactions and plant-dependent microbial metabolisms in the endosphere. Finally, this book chapter highlights essential research needs and gaps to substantiate the necessity to extend SIP-based approaches for prospective studies within the active and multifaceted plant–microbe ecosystem.