Isolation and characterisation of novel nematophagous fungi from eggs of the cereal cyst nematode Heterodera filipjevi and identifying their secondary metabolites with nematicidal activity Dissertation Zur Erlangung des Akademischen Grades eines Doktors der Agrarwissenschaften (Dr. agr.) Eingereicht am Fachbereich Ökologische Agrarwissenschaften der Universität Kassel Von Samad Ashrafi February 2018 Samad Ashrafi (2018) Isolation and characterisation of novel nematophagous fungi from eggs of the cereal cyst nematode Heterodera filipjevi and identifying their secondary metabolites with nematicidal activity A thesis in Biological control: University of Kassel Defence date: 17.04.2018 Place: Witzenhausen, Germany Supervisors: Prof. Dr. Maria R. Finckh (University of Kassel) Dr. Wolfgang Maier (Julius Kühn-Institut — Federal Research Center for Cultivated Plants) For Saleh Table of Contents List of Tables ........................................................................................................................................... vii List of Figures ......................................................................................................................................... viii Chapter 1. General Introduction ....................................................................................................... 1 1.1 Cereal cyst nematodes ............................................................................................................ 1 1.2 Biocontrol of nematodes ......................................................................................................... 3 1.3 Nematophagous fungi ............................................................................................................. 4 1.3.1 Nematode-trapping fungi ................................................................................................ 5 1.3.2 Endoparasitic fungi .......................................................................................................... 6 1.3.3 Egg-parasitic fungi ........................................................................................................... 7 1.4 Suppression of PPNs with nematophagous fungi ................................................................... 8 1.5 Nematophagous fungi, a source of nematicidal metabolites ............................................... 11 1.6 Introduction to the project.................................................................................................... 12 1.7 Research rationale and objectives of the project ................................................................. 15 1.8 Refernces ............................................................................................................................... 17 Chapter 2. Ijuhya vitellina sp. nov., a novel source for chaetoglobosin A, is a destructive parasite of the cereal cyst nematode Heterodera filipjevi .......................................................... 22 2.1 Introduction ........................................................................................................................... 23 2.2 Material and methods ........................................................................................................... 24 2.2.1 Nematode collection and materials examined ............................................................. 24 2.2.2 Ethics statement ............................................................................................................ 25 2.2.3 Fungal isolation from field collected samples ............................................................... 25 2.2.4 Pure culture based studies ............................................................................................ 26 2.2.5 Pathogenicity test and Koch’s postulate ....................................................................... 26 2.2.6 Light and scanning electron microscopy ....................................................................... 27 2.2.7 DNA extraction .............................................................................................................. 27 2.2.8 PCR amplification and sequencing ................................................................................ 28 2.2.9 Alignment and phylogenetic reconstruction ................................................................. 28 2.2.10 Nomenclature ................................................................................................................ 37 2.2.11 Metabolite profiling....................................................................................................... 37 2.3 Results ................................................................................................................................... 38 2.3.1 Fungal isolation ............................................................................................................. 38 2.3.2 Molecular phylogenetic studies .................................................................................... 39 2.3.3 Taxonomy ...................................................................................................................... 42 2.3.4 In vitro parasitism on nematode eggs and Koch’s postulates ....................................... 45 iv 2.3.5 Metabolite profiling....................................................................................................... 48 2.4 Discussion .............................................................................................................................. 49 2.4.1 Phylogenetic analyses and systematic implications ...................................................... 49 2.4.2 Parasitism on nematode cysts and eggs ....................................................................... 50 2.4.3 Metabolite profiling....................................................................................................... 51 2.5 Appendix ................................................................................................................................ 53 2.6 References ............................................................................................................................. 54 2.7 Supporting information ......................................................................................................... 60 Chapter 3. Monocillium gamsii sp. nov. and Monocillium bulbillosum: two nematode-associated fungi parasitising the eggs of Heterodera filipjevi ......................................................... 67 3.1 Introduction ........................................................................................................................... 68 3.2 Material and methods ........................................................................................................... 69 3.2.1 Sample collection and materials examined ................................................................... 69 3.2.2 Cultural studies .............................................................................................................. 70 3.2.3 Pathogenicity tests against H. filipjevi ........................................................................... 70 3.2.4 Microscopy .................................................................................................................... 71 3.2.5 Molecular phylogenetic studies .................................................................................... 71 3.3 Results ................................................................................................................................... 74 3.3.1 Sample collection and fungal isolation .......................................................................... 74 3.3.2 Sequence comparison and phylogenetic inference ...................................................... 75 3.3.3 Taxonomy ...................................................................................................................... 76 3.3.4 Development of M. gamsii in nematode eggs in vitro .................................................. 79 3.3.5 Parasitism of M. bulbillosum towards H. filipjevi .......................................................... 81 3.4 Discussion .............................................................................................................................. 81 3.5 References ............................................................................................................................. 84 Chapter 4. Inhabiting plant roots, nematodes and truffles — Polyphilus nom. prov., a new helotialean genus with two globally distributed species .............................................. 87 4.1 Introduction ........................................................................................................................... 88 4.2 Material and methods ........................................................................................................... 90 4.2.1 Origin of isolates ............................................................................................................ 90 4.2.2 Fungal isolation from nematode eggs ........................................................................... 90 4.2.3 Sporulation and morphology of isolates ....................................................................... 90 4.2.4 DNA extraction and amplification ................................................................................. 92 4.2.5 Phylogenetic analyses ..................................................................................................