Interactions between invertebrate and mycotoxin-producing fungi Dissertation to obtain the Ph. D. degree in the International Ph. D. Program for Agricultural Sciences in Göttingen (IPAG) at the Faculty of Agricultural Sciences, Georg-August-University Göttingen, Germany Presented by Yang Xu Born in China Göttingen, 05.2018 D7 (this is the number of our faculty it’s important for the university library) 1. Name of supervisor: Prof. Dr. Petr Karlovsky 2. Name of co-supervisor: Prof. Dr. Stefan Vidal Date of dissertation: 07.09.2018 Contents CHAPTER 1: GENERAL INTRODUCTION.................................................................. 1 1. Fungi as invertebrate food source .............................................................................................................. 1 1.1. Invertebrate diet ..................................................................................................................................... 1 1.2. Effect of predation on fungal biomass .................................................................................................. 1 2. Mutualism between fungus and invertebrate ............................................................................................ 2 3. Entomopathogenic fungi ............................................................................................................................. 2 4. Fungi can repel invertebrates ..................................................................................................................... 3 5. Animals as vectors for fungal spores ......................................................................................................... 3 6. Invertebrate food preference ...................................................................................................................... 3 7. Fungal defense against predation ............................................................................................................... 4 8. Molecules mediates interactions between fungi and invertebrates ......................................................... 5 8.1. Secondary metabolites ............................................................................................................................ 5 8.2. Proteins .................................................................................................................................................... 6 9. Methods used to investigate insect-fungal interactions ............................................................................ 7 9.1. Microscopy .............................................................................................................................................. 7 9.2. Food preference and fitness of animals................................................................................................. 7 9.3. Defense analysis of grazed mycelium .................................................................................................... 7 9.4. Analysis of microorganisms associated with invertebrates ................................................................. 7 10. Aim of this study ........................................................................................................................................ 8 11. References .................................................................................................................................................. 8 CHAPTER 2: EFFECT OF ANIMAL PREDATION ON THE BIOMASS OF MYCOTOXIN-PRODUCING FUNGI............................................................................ 16 Abstract .......................................................................................................................................................... 16 1. Introduction ............................................................................................................................................... 17 2. Material and methods ............................................................................................................................... 17 2.1. Fungal strains and animals .................................................................................................................. 18 2.2. Fungal exposure to collembolans ........................................................................................................ 18 2.3. Preparation of DNA standards for the determination of A. ochraceus, A. nidulans and P. viridicatum biomass ....................................................................................................................................... 18 2.4. DNA extraction of grazed and non-grazed fungal mycelia ............................................................... 19 2.5. Quantification of fungal DNA by qPCR ............................................................................................. 19 2.6. Statistical ............................................................................................................................................... 20 3. Results ........................................................................................................................................................ 20 3.1. Standard curve of each fungal strain for real-time PCR .................................................................. 20 3.2. Fungal weight after animal predation ................................................................................................ 21 3.3. DNA quality .......................................................................................................................................... 21 3.4. Fungal biomass assessment by real-time PCR ................................................................................... 23 4. Discussion ................................................................................................................................................... 25 5. References .................................................................................................................................................. 27 6. Extended data ............................................................................................................................................ 30 CHAPTER 3: INVESTIGATION OF INTERACTIONS BETWEEN FUSARIUM GRAMINEARUM, FUSARIUM VERTICILLIOIDES AND FOLSOMIA CANDIDA (COLLEMBOLA) ............................................................................................................. 32 Abstract .......................................................................................................................................................... 32 1. Introduction ............................................................................................................................................... 33 2. Material and Method ................................................................................................................................ 34 2.1. Test organisms ...................................................................................................................................... 34 2.2. Food preference of F. candida ............................................................................................................. 34 2.3. Quantitation of consumed mycelium .................................................................................................. 35 2.3.1. Food preparation ............................................................................................................................. 35 2.3.2. DNA extraction ................................................................................................................................ 36 2.3.3. Quantitative real-time PCR ............................................................................................................ 36 3. Results ........................................................................................................................................................ 36 3.1. F. candida food preference between F. verticillioides and F. graminearum ..................................... 36 3.2. Food choice of F. candida offered F. verticillioides and F. graminearum mutants impared in secondary metabolite synthesis ..................................................................................................................... 37 3.3. Food preference between F. graminearum, F. verticillioides, and their mixed culture ................... 38 3.4. Comparison between food selection and food preference ................................................................. 39 4. Discussion ................................................................................................................................................... 39 5. References .................................................................................................................................................. 41 CHAPTER 4: NONTOXIC NAPHTHOPYRONE PIGMENT PROTECTS FILAMENTOUS FUNGI FROM A WIDE RANGE OF ANIMAL PREDATORS ... 44 1. Main text .................................................................................................................................................... 44 2. Materials and Methods ............................................................................................................................. 53 2.1. Animals .................................................................................................................................................