The association between overlooked microbial eukaryotes and plant holobiont: functionality and diversity Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Alfredo Mari aus Pescara, Italien Köln, 2018 II III Die vorliegende Arbeit wurde am Max-Planck-Institut für Pflanzenzüchtungsforschung in Köln in der Abteilung für Pflanze-Mikroben Interaktionen (Direktor: Prof. Dr. Paul Schulze-Lefert) angefertigt. Berichterstatter: Prof. Dr. Gunther Döhlemann Prof. Dr. Eric Kemen Schriftführer: Dr. Korbinian Schneeberger Prüfungsvorsitzender: Prof. Dr. Achim Tresch Tag der Disputation: 23.07.2018 IV V “I got power, poison, pain and joy inside my DNA, I got hustle though, ambition flow inside my DNA” Kendrick Lamar, Pulitzer prize 2017. DNA VI VII Table of Contents Publications: ................................................................................................................. IV List of tables ................................................................................................................... V List of Figures ................................................................................................................ V List of Abbreviations and Acronyms .......................................................................... VIII Abstract: ................................................................................................................... XI Zusammenfassung: ................................................................................................. XIII 1. Introduction ........................................................................................................ 1 1.1 Plant microbiome: ecological importance and implications for plant health ................. 1 1.2 The incomplete picture: ecological insights from overlooked microbial eukaryotes can reveal the missing piece of the puzzle ............................................................................... 4 1.2.1 Biogeography of overlooked microbial eukaryotes: which are the ecological drivers of microbial populations? ................................................................................. 6 1.2.2 Plant holobiont encompasses several primary producers: autotrophic organisms associated with plants ................................................................................................... 8 1.2.3 Micropredators: heterotrophic microbes shaping the overall diversity of plant associated microbes .................................................................................................... 10 1.2.4 Keystone species are not necessarily abundant: the role of rare taxa in microbial assembly ..................................................................................................................... 13 1.3 Methods to study microbiome: high throughput amplicon sequencing ....................... 14 1.3.1 Network analysis majorly strengthen ecological analysis ................................... 15 1.4 Aim of the thesis and experimental setup .................................................................. 16 2. Results .............................................................................................................. 20 2.1 Ecological relevance of microbial eukaryotes in the plant holobiont ........................... 20 2.1.1 Sequencing taxonomical markers from microbial eukaryotes: blocking oligos substantially increase sequencing resolution ............................................................... 20 2.1.2 Shedding light on leaf-associated overlooked microbial eukaryotes: more than 14 subdivisions compose the mosaic .......................................................................... 22 2.1.2.1 Connected microbes: overlooked microbial eukaryotes as a fundamental part of microbial food webs ....................................................................................................... 24 2.1.3 Biogeography of leaf microbiome: habitat and province shape microbial diversity depending on the taxonomic group ........................................................................... 26 2.1.4 Fitting overlooked microbial eukaryotes and environmental features in microbial networks provides insights on biotic interactions on the phyllosphere ...................... 28 2.1.4.1 Chlorophyta and Metazoa are key nodes in microbial networks ....................... 28 2.1.4.2 Bracteacoccus sp. hampers connections towards potential pathogens such as Pseudomonas viridiflava .................................................................................................. 32 2.1.4.2.1 A new pipeline for solid and quantitative network comparison unravels the lack of correlation between Brac+ and Brac- network ................................... 33 2.2 Investigating the role of producers in terrestrial ecosystems ....................................... 37 2.2.1 Overlooked microbial eukaryotes shaping microbial diversity: Bracteacoccus sp., is a keyplayer, together with Metazoa, Lobosa and Ciliophora ..................................... 37 I 2.2.2 Chloroplastic DNA confirms presence of algal reads in the endophytic compartment .............................................................................................................. 40 2.2.3 Endophytic lichens belong to Lecanorales order .............................................. 41 2.2.3.1 Lichenising fungi impact on bacteria diversity only when co-occurring with Algae .............................................................................................................................. 43 2.2.3.2 Dissecting the role of lichen on the leaf. Time course over different ecotypes reveals fluctuations across time, but not across genotype ............................................... 43 2.2.4 Mechanisms underlying algae role in leaf ecosystem do not involve differential sugar productions ....................................................................................................... 46 2.3 Dissecting the role of heterotrophs and amoeboid organisms: predators on the leaf surface ........................................................................................................................... 48 2.3.1 Successions of consumers: different time for different predators ...................... 48 2.4 United they stand: rare taxa play an important role in shaping the diversity, but only if taken together ................................................................................................................ 50 3. Discussion ......................................................................................................... 53 3.1 Methodological advances in amplicon sequencing and downstream data analysis ....... 55 3.1.1 Blocking oligonucleotides enhance sequencing resolution and improve low abundant taxa’s detectability ...................................................................................... 55 3.1.2 A new network comparison pipeline provides customizable, modular and solid statistics for ecological studies .................................................................................... 56 3.2 Overlooked microbial eukaryotes biogeography appears to be shaped by microhabitat factors rather than by geographical province ................................................................... 58 3.2.1 Overlooked microbial eukaryotes and microhabitat: biotic interactions are not influenced by province related variables, underpinning a bond with the biome variable ................................................................................................................................... 61 3.3 The leaf in the leaf: microalgae in both compartments reveal unexpected microbial dynamics ....................................................................................................................... 63 3.3.1 Phycosphere in the phyllosphere, microalgae shape microbial interactions by connecting to the key hubs, likely through biofilm .................................................... 64 3.3.2 Bracteacoccus sp may be the main player in the biofilm niche ............................. 65 3.4 The fungal connection: Lichens ................................................................................ 66 3.4.1 Lichens appear in both endophytic and epiphytic compartment, and they impact bacterial diversity on the leaf ...................................................................................... 66 3.4.2 The key role of lichens in the phyllosphere microbiome is likely dependent on microhabitat conditions and may involve different phycobionts. Host genotype is not directly involved ......................................................................................................... 68 3.5 Consumers and grazing: connected through different timing, Lobosa, Conosa, Ciliophora and Metazoa exert a striking effect on microbial assembly .............................. 71 3.5.1 Predator and prey revisited: when the prey can become a symbiont ................. 73 3.5.2 Cercozoa: the big absent ................................................................................... 74 3.6 Rare taxa guarantee microbiome resilience, however, they only have effect if taken together ......................................................................................................................... 74 3.7 Conclusions and further perspectives .......................................................................