Diversity, phylogeny, characterization and diagnostics of root-knot and lesion nematodes Toon Janssen Promotors: Prof. Dr. Wim Bert Prof. Dr. Gerrit Karssen Thesis submitted to obtain the degree of doctor in Sciences, Biology Proefschrift voorgelegd tot het bekomen van de graad van doctor in de Wetenschappen, Biologie 1 Table of contents Acknowledgements Chapter 1: general introduction 1 Organisms under study: plant-parasitic nematodes .................................................... 11 1.1 Pratylenchus: root-lesion nematodes ..................................................................................... 13 1.2 Meloidogyne: root-knot nematodes ....................................................................................... 15 2 Economic importance ..................................................................................................... 17 3 Identification of plant-parasitic nematodes .................................................................. 19 4 Variability in reproduction strategies and genome evolution ..................................... 22 5 Aims .................................................................................................................................. 24 6 Outline of this study ........................................................................................................ 25 Chapter 2: Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution. 1 Abstract ............................................................................................................................ 27 2 Introduction ..................................................................................................................... 27 3 Material and methods ..................................................................................................... 30 3.1 Collection of populations, morphological identification and culturing ................................. 30 3.2 Isozyme analysis .................................................................................................................... 34 3.3 Mitochondrial DNA analysis ................................................................................................. 34 4 Results .............................................................................................................................. 36 4.1 Sampling and isozyme electrophoresis ................................................................................. 36 4.2 Identification of polymorphic sites within the mitochondrial genome .................................. 39 4.2.1 Meloidogyne enterolobii ................................................................................................ 39 4.2.2 MIG lineages ................................................................................................................. 39 4.3 Multi-gene haplotype network .............................................................................................. 43 5 Discussion ......................................................................................................................... 45 6 Supplementary information Chapter 2 ......................................................................... 51 2 Chapter 3: Integrative taxonomy of root-knot nematodes reveals multiple independent origins of mitotic parthenogenesis. 1 Abstract ............................................................................................................................ 58 2 Introduction ..................................................................................................................... 59 3 Material and methods ..................................................................................................... 62 3.1 Collection of populations, culturing and host-range test ....................................................... 62 3.2 Morphological characterization ............................................................................................. 63 3.3 Isozyme analysis .................................................................................................................... 64 3.4 DNA extraction, PCR amplification and sequencing ............................................................ 64 3.5 Sequence analysis .................................................................................................................. 65 3.6 Cytological staining ............................................................................................................... 66 4 Results and discussion .................................................................................................... 66 4.1 Morphological identification of coffee root-knot nematodes from Tanzania ....................... 66 4.1.1 Females .......................................................................................................................... 66 4.1.2 Second-stage juveniles .................................................................................................. 71 4.1.3 Males ............................................................................................................................. 74 4.1.4 Conclusion of morphological identification .................................................................. 78 4.2 Redescription of Meloidogyne africana (Table 4, Fig 1, 2, 3 and 4) .................................... 79 4.3 Phylogenetic analysis, molecular diagnosis and evolutionary morphology .......................... 82 4.3.1 Phylogenetic analyses .................................................................................................... 82 4.3.2 Evolutionary morphology of the perineal pattern and protuberance ............................. 87 4.3.3 Evolutionary morphology of the female gonoduct ........................................................ 87 4.4 Isozyme electrophoresis ........................................................................................................ 88 4.5 Evolution of reproduction and oogenesis .............................................................................. 89 4.5.1 Meloidogyne javanica ................................................................................................... 89 4.5.2 Meloidogyne africana .................................................................................................... 90 4.5.3 Meloidogyne ardenensis ................................................................................................ 90 4.5.4 Meloidogyne mali .......................................................................................................... 91 4.5.5 Evolution of reproduction and oogenesis in root-knot nematodes ................................ 92 4.6 Bionomics .............................................................................................................................. 96 5 Acknowledgements ......................................................................................................... 97 3 6 Supplementary information Chapter 3 ......................................................................... 98 Chapter 4: Molecular characterization and species delimiting of plant-parasitic nematodes of the genus Pratylenchus from the Penetrans group (Nematoda: Pratylenchidae). 1 Abstract .......................................................................................................................... 100 2 Introduction ................................................................................................................... 100 3 Material and methods ................................................................................................... 103 3.1 Nematode populations ......................................................................................................... 103 3.2 Morphological study ............................................................................................................ 103 3.3 DNA extraction, PCR amplification and sequencing .......................................................... 104 3.4 Sequence and phylogenetic analyses ................................................................................... 112 3.5 Species delimitation ............................................................................................................ 112 4 Results and taxonomic notes ........................................................................................ 113 4.1 Dataset and taxonomic notes ............................................................................................... 113 4.1.1 Pratylenchus arlingtoni Handoo, Carta & Skantar, 2001............................................ 114 4.1.2 Pratylenchus brachyurus (Godfrey 1929) Filipjev & Schuurmans Stekhoven, 1941 . 115 4.1.3 Pratylenchus convallariae Seinhorst, 1959 ................................................................. 115 4.1.4 Pratylenchus dunensis de la Pena, Moens, van Aelst & Karssen, 2006 ...................... 118 4.1.5 Pratylenchus fallax Seinhorst 1968 ............................................................................. 119 4.1.6 Pratylenchus penetrans (Cobb, 1917) Filipjev & Schuurmans Stekhoven, 1941 ....... 119 4.1.7 Pratylenchus pinguicaudatus Corbett, 1969 ............................................................... 120 4.1.8 Pratylenchus sp. 1 ......................................................................................................