PhD School of Chemistry, Biochemistry and Ecology of Pesticide (CBEA) DOCTORAL DISSERTATION Tagging biocontrol Streptomyces to study lettuce colonization Xiaoyulong Chen To be presented with the permission of the examining board, 28th cycle of the CBEA PhD school, for public examination at Aula Magna C03 (Room C03), Faculty of Agricultural and food sciences, University of Milan, on 17.12.2015. Cover figure: Colonization of lettuce root by Streptomyces cyaneus ZEA17I, examined by confocal laser scanning microscopy. Supervisor: Professor Paolo Cortesi Department of Food, Environmental, and Nutritional Sciences University of Milan, Italy Co-supervisor: Professor Marco Saracchi Department of Food, Environmental, and Nutritional Sciences University of Milan, Italy Coordinator: Professor Daniele Daffonchio Department of Food, Environmental, and Nutritional Sciences University of Milan, Italy Examination board (ordered by first letter of the family name): 1. Professor Paolo Bertolini Department of Agricultural Sciences University of Bologna, Italy 2. Dr. Jonas Bengtsson Department of Zoology Stockholm University, Sweden 3. Professor Daniele Daffonchio Department of Food, Environmental, and Nutritional Sciences University of Milan, Italy 4. Professor George Tsiamis Dep. of Environmental and Nutritional Resources Management University of Patras, Greece 5. Dr. Laura Polito Institute of Molecular Science and Technology, CNR, Italy Contents List of original publications....................................................................................................... 1 List of conferences attended ...................................................................................................... 2 List of awards ............................................................................................................................ 3 Invitation for peer-review .......................................................................................................... 3 Abbreviations ............................................................................................................................ 4 List of figures ............................................................................................................................ 5 List of tables .............................................................................................................................. 6 Abstract ..................................................................................................................................... 7 Introduction ............................................................................................................................... 9 Lettuce ................................................................................................................................... 9 Towards sustainable agriculture: the application of biological control agents .................... 14 Streptomyces spp. in nature, and their functions in agriculture ........................................... 17 The crucial role of plant colonization by microorganisms in biocontrol mechanisms ........ 20 Antibiotic resistance markers as fundamental tools to study microbe colonization dynamics ............................................................................................................................................. 24 EGFP marker as a convenient approach to visualize colonization patterns of beneficial microbes .............................................................................................................................. 25 FISH, a cytogenetic and useful technique to study plant-microbe interactions................... 27 Aim of the study ...................................................................................................................... 29 Materials and methods ............................................................................................................. 31 1. Transformation of the Streptomyces strains with the pIJ8641 construct ..................... 31 1.1 Conjugation ............................................................................................................. 31 1.2 PCR amplification of the EGFP-transgene .............................................................. 32 1.3 Microscopic observation of EGFP expression ........................................................ 32 2. Impact of the transformation on fitness of Streptomyces strains ................................. 33 2.1 Antagonistic activity against Sclerotinia sclerotiorum ........................................ 33 2.2 Sporulation .......................................................................................................... 34 2.3 Mycelium biomass growth .................................................................................. 34 2.4 Production of secondary metabolites ................................................................... 35 3. Lettuce colonization dynamics by two tagged Streptomyces strains ........................... 36 3.1 Lettuce bulk soil colonization.............................................................................. 37 3.2 Lettuce rhizosphere colonization ......................................................................... 37 3.3 Lettuce inner-root colonization ........................................................................... 38 4. CLSM observation of lettuce root colonization by S. cyaneus EGFP-ZEA17I........... 38 5. CLSM observation in combination with fluorescence in situ hybridization (FISH) ... 40 6. Statistical analysis ....................................................................................................... 41 Results ..................................................................................................................................... 42 1. Transformation of Streptomyces strains with the pIJ8641 construct ........................... 42 1.1 Conjugation ............................................................................................................. 42 1.2 PCR amplification of the EGFP-transgene .............................................................. 43 1.3 Microscopic observation of EGFP expression ........................................................ 43 2. Impact of the transformation on fitness of Streptomyces strains ................................. 45 2.1 Antagonistic activity ............................................................................................ 45 2.2 Sporulation .......................................................................................................... 46 2.3 Mycelium biomass growth .................................................................................. 47 2.4 Production of secondary metabolites ................................................................... 48 3. Lettuce colonization dynamics by two tagged Streptomyces strains ........................... 50 3.1 Bulk soil colonization .......................................................................................... 50 3.2 Lettuce rhizosphere colonization ......................................................................... 51 3.3 Rhizosphere/bulk soil proportion ........................................................................ 53 3.4 Lettuce inner-root colonization ........................................................................... 53 4. CLSM observation of lettuce colonization by EGFP-ZEA17I .................................... 54 4.1 One-week-old plant ............................................................................................. 54 4.2 Two-week-old plant ............................................................................................. 56 5. CLSM observation of lettuce colonization by EGFP-ZEA17I in combination with FISH .................................................................................................................................... 57 5.1 One-week-old plant ............................................................................................. 57 5.2 Two-week-old plant ............................................................................................. 59 Discussion ............................................................................................................................... 61 Conclusions: ............................................................................................................................ 68 Acknowledgements ................................................................................................................. 69 References ............................................................................................................................... 72 Appendix ................................................................................................................................. 89 Publication No.1: ................................................................................................................. 89 Publication No.2: ................................................................................................................. 89 Publication No.3: ................................................................................................................. 89 Publication No.4: ................................................................................................................. 89 Award No.1: .......................................................................................................................