Pollen Associated Microbiome and Its Relationship to Pollution and Allergens
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Pollen associated microbiome and its relationship to pollution and allergens Andrea Obersteiner Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Department für Umweltwissenschaften Abteilung Mikroben-Pflanzen-Interaktion Dissertation der Fakultät für Biologie der Ludwig-Maximilian-Universität München zur Erlangung des akademischen Grad eines Doktor der Naturwissenschaften (Dr. rer. nat.) Erstgutachter: Prof. Dr. Anton Hartmann Zweitgutachter: Prof. Dr. Kirsten Jung Eingereicht am: 09.08.2016 Tag der mündlichen Prüfung: 24.01.2017 LIST OF PUBLICATIONS Obersteiner A, Gilles S, Frank U, Beck I, Häring F, Ernst D et al. (2016) Pollen- Associated Microbiome Correlates with Pollution Parameters and the Allergenicity of Pollen. PLoS ONE 11(2): e0149545. doi:10.1371/journal.pone.0149545 Obersteiner A*, Rauer D*, Reiger M, Gilles S, et al. (in preparation) The role of pollen-associated phytopathogenic bacteria in allergenic immune response. *Co-Authors POSTERS AND ORAL PRESENTATIONS 08/2014 Oral presentation on the ISME15 conference (International Symposium of Microbiological Ecology) in Seoul, South Korea 10/2014 Poster presentation on the DAK (Deutscher Allergie Kongress) in Wiesbaden, Germany 03/2016 Poster presentation on the DGAKI (Deutsche Gesellschaft für Allergologie und klinische Immunologie) in Mainz, Germany TABLE OF CONTENTS TABLE OF CONTENTS LIST OF ABBREVIATIONS .......................................................................................................... 5 ABSTRACT ................................................................................................................................. 7 ZUSAMMENFASSUNG .............................................................................................................. 9 1 INTRODUCTION ............................................................................................................... 11 1.1 The ecology and structure of pollen grains .................................................................. 12 1.2 Abiotic and biotic plant interactions ............................................................................ 15 1.3 Plant defense mechanisms ........................................................................................... 18 1.4 Stress-induced allergen-expression ............................................................................. 19 1.5 Pollen impact on human health ................................................................................... 20 1.6 Modern methods of molecular microbiology .............................................................. 21 1.7 An overview of the thesis ............................................................................................. 24 2 MATERIAL AND METHODS .............................................................................................. 25 2.1 Sample collection and preparation .............................................................................. 25 2.1.1 Preparation of culture stocks ............................................................................. 25 2.1.2 Preparation for community analysis .................................................................. 26 2.2 Cultivation of pollen-associated microbes ................................................................... 26 2.2.1 Selection of pollen samples for cultivation ........................................................ 26 2.2.2 Preparation of media for cultivation .................................................................. 27 2.2.3 Conditions of cultivation of microbes ................................................................ 29 2.2.4 Differentiation of isolated pure cultures by Restriction Fragment Length Polymorphism (RFLP) ......................................................................................... 29 2.2.5 Horizontal agarose gel electrophoresis .............................................................. 31 2.2.6 Identification of microbial isolates from pollen ................................................. 31 2.3 Molecular-biological standard techniques ................................................................... 32 2.3.1 Extraction of chromosomal DNA from isolated pure cultures ........................... 32 2.3.2 Extraction of microbial community-DNA ........................................................... 32 2.3.3 Purification of isolated DNA ............................................................................... 33 2.3.4 Determination of DNA-concentrations .............................................................. 33 2.4 Bacterial community determined by amplicon sequencing ........................................ 33 2.4.1 Sample selection ................................................................................................ 34 2.4.2 Separation of plastid DNA performing 16S-PCR with 799f/1492r ..................... 36 2.4.3 Nested-PCR ......................................................................................................... 38 2.4.4 Quantification of DNA with PicoGreen .............................................................. 38 2.4.5 Indexing PCR ....................................................................................................... 39 2.4.6 Equimolar pooling .............................................................................................. 40 1 TABLE OF CONTENTS 2.4.7 Illumina BaseSpace Data Analysis ...................................................................... 41 2.5 Fluorescence in situ Hybridization (FISH) ..................................................................... 41 2.5.1 Fixation in paraformaldehyde (PFA) .................................................................. 41 2.5.2 Hybridization ...................................................................................................... 42 2.5.3 Confocal Laser Scanning- and Epifluorescence Microscopy .............................. 44 2.6 Environmental and allergenicity related parameter .................................................... 44 2.7 Terminal restriction fragment length polymorphism (tRFLP) ...................................... 45 2.7.1 Amplification of bacterial 16S-rDNA fragments and fungal internal .................... transcribed spacer region (ITS 1-2) ................................................................... 45 2.7.2 Enzymatic restriction digest ............................................................................... 46 2.7.3 tRFLP data-analysis ............................................................................................. 47 2.7.4 Statistical analysis of tRFLP-data ........................................................................ 47 2.7.5 Exclusion of plastid DNA from tRFLP-data ......................................................... 49 2.7.5.1 Determining the length of plastid terminal restricted fragment ................... 49 2.7.5.2 Sequencing of plastid fragment after cloning ................................................ 49 3 RESULTS .......................................................................................................................... 51 3.1 Identification of cultivable pollen-associated microbes .............................................. 51 3.1.1 Fungal strains isolated from timothy grass pollen ............................................. 51 3.1.2 Fungal strains isolated from birch pollen ........................................................... 52 3.1.3 Bacterial strains isolated from timothy grass pollen ......................................... 54 3.1.4 Bacterial strains isolated from birch pollen ....................................................... 57 3.2 Diversity analysis of microbial community ................................................................... 59 3.2.1 Fungal tRFLP-pattern analysis ............................................................................ 59 3.2.2 Fungal α-diversity based on tRFLP patterns ....................................................... 61 3.2.3 Bacterial tRFLP-pattern analysis ......................................................................... 63 3.2.4 Bacterial α-diversity based on tRFLP-patterns ................................................... 65 3.2.5 Bacterial community identification by 16S-rDNA amplicon sequencing ........... 67 3.2.5.1 Bacterial composition on timothy grass pollen .............................................. 67 3.2.5.2 Bacterial composition on birch pollen ............................................................ 70 3.3 Fluorescence in situ Hybridization (FISH) ..................................................................... 74 3.3.1 -Proteobacteria on timothy grass pollen .......................................................... 74 3.3.2 α-Proteobacteria on birch pollen ....................................................................... 76 3.4 Air pollution and pollen-associated microbial composition ........................................ 79 3.4.1 Comparison of microbial patterns on pollen from rural and urban locations ... 79 3.4.2 Pollution parameters correlated to bacterial diversity on birch pollen ............ 80 3.4.3 Pollution parameters correlated to fungal diversity on birch pollen ................ 82 2 TABLE OF CONTENTS 3.4.4 Urbanization Index correlated to bacterial and fungal diversity on timothy grass pollen ........................................................................................... 82 3.5 Allergen-related