Functional Roles of Cercozoa in the Rhizosphere and Phyllosphere of Plants Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Sebastian Flues aus Friedrichshafen Köln, 2018 Berichterstatter: Prof. Dr. Michael Bonkowski Prof. Dr. Hartmut Arndt Vorsitz der Prüfung: Prof. Dr. Frank Schäbitz Beisitzer: Dr. Kenneth Dumack Tag der mündlichen Prüfung: 15.12.2017 Table of Contents Table of Contents Summary ............................................................................................... 6 Zusammenfassung ............................................................................... 8 General Introduction .......................................................................... 10 Protists ................................................................................................................... 10 Functional roles of protists in soil ........................................................................... 12 Protists in the rhizosphere of plants ....................................................................... 13 Leaf-associated protists in the phyllosphere of plants ............................................ 14 Cercozoa ................................................................................................................ 16 Cercomonadida - the dominant terrestrial amoeboflagellates........................................ 17 Testacea in the Cercozoa ............................................................................................. 17 Aims ....................................................................................................................... 18 Chapter 1 - Diversity of Cercomonad Species in the Phyllosphere and Rhizosphere of Different Plant Species with a Description of Neocercomonas epiphylla (Cercozoa, Rhizaria) a Leaf-Associated Protist .................................................................................................. 22 Abstract .................................................................................................................. 22 Introduction ............................................................................................................ 23 Material and Methods ............................................................................................. 25 Results ................................................................................................................... 28 Discussion .............................................................................................................. 40 Supporting Information ........................................................................................... 43 Chapter 2 - Grazing of leaf-associated Cercomonads (Protists: Rhizaria: Cercozoa) structures bacterial community composition and function ........................................................................................ 47 Abstract .................................................................................................................. 47 Introduction ............................................................................................................ 48 Material and Methods ............................................................................................. 50 3 Table of Contents Results ................................................................................................................... 55 Discussion .............................................................................................................. 65 Supporting Information ........................................................................................... 69 Chapter 3 - Spatial and Temporal Dynamics between Leaf-Associated Cercomonad Cercozoa and Phyllosphere Bacteria on Bean Leaves (Phaseolus vulgaris)............................................... 76 Introduction ............................................................................................................ 76 Material and Methods ............................................................................................. 78 Results and Discussion .......................................................................................... 81 Conclusion ............................................................................................................. 92 Chapter 4 - Rhogostomidae (Cercozoa) from soils, roots and plant leaves (Arabidopsis thaliana): Description of Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov. .................................................... 94 Abstract .................................................................................................................. 94 Introduction ............................................................................................................ 95 Material and Methods ............................................................................................. 97 Results ................................................................................................................. 100 Discussion ............................................................................................................ 109 Supporting Information ......................................................................................... 111 General Discussion .......................................................................... 112 Diversity of leaf-associated Cercozoa .................................................................. 113 Functional roles of leaf-associated Cercozoa ...................................................... 114 Grazing structures bacterial community composition and interaction .......................... 115 Grazing alters bacterial community function ................................................................ 116 Leaf-associated Cercozoa are more than bacterial feeders ........................................ 117 Dynamics between Cercozoa and bacteria on leaves.......................................... 117 Diversity and functional roles of Cercozoa in the rhizosphere .............................. 119 Leaf-associated protists and their relevance for phytobiome research ................ 120 References ........................................................................................ 122 4 Table of Contents Acknowledgements .......................................................................... 149 Subpublications and Record of Achievement ................................ 150 Erklärung gemäß § 4 Absatz 1 Punkt 9 der Prüfungsordnung ...... 152 5 Summary Summary Protists are a group of highly diverse unicellular eukaryotic organisms. They are globally distributed and inhabit all types of terrestrial and aquatic habitats. Protists represent an enormous functional diversity and occupy various ecological niches as bacterivores, fungivores, algivores, predators, primary producers, saprotrophs or parasites of plants and animals. Among protists, the Cercozoa are one of the most diverse, speciose and ecologically important of all protist phyla. Protists in terrestrial systems often show an association to plants and they are well-known predators on plant surfaces. However, a comprehensive understanding about the distribution and functions of plant-associated protists in the rhizosphere (i.e. belowground compartments of plants) and the phyllosphere (i.e. aboveground compartments of plants, mainly leaves) is lacking. Therefore this thesis aims to increase the knowledge on the diversity and functional roles of plant-associated Cercozoa. In the first chapter several cercomonad Cercozoa strains were isolated from the phyllosphere and rhizosphere of plants from three functional groups. Their potential phyllosphere and rhizosphere as well as plant specificity was investigated and revealed that cercomonad communities show a deterministic assembly in the above- and belowground compartments of plants. During the course of this study, three novel cercomonad species were described and ten new cercomonad genotypes reported. This indicates that cercozoan taxa preferentially associated with the phyllosphere exist and that Cercozoa diversity is far from being completely revealed. The second chapter aims at deciphering the feeding preferences of leaf-associated cercomonads and their predation effects on the composition, function and interaction of phyllosphere bacterial communities. Predation-induced shifts in bacterial community composition could be linked to phenotypic protist traits and we showed that leaf-associated cercomonads significantly structured bacterial community composition which led to an altered interaction pattern among bacterial taxa. This study further demonstrated that cercomonad predation can have significant impact on the physiological function of bacterial communities. The third chapter of this thesis aimed to reveal the spatial and temporal dynamics between leaf-associated cercomonad Cercozoa and phyllosphere bacteria on leaves. We could prove that leaf-associated cercomonads are active and feeding on bacterial cells on the leaf surface and confirm the activity of protists on plant surfaces is closely connected to moisture on leaves. The last chapter aimed to increase the knowledge on the ecology and function of plant-associated cercozoan testate amoebae. Four different strains of Rhogostoma spp. were isolated from Arabidopsis leaves, agricultural soil and rhizosphere soil of Ocimum basilicum and Nicotiana sp. Detailed