Host range and genetic characterization of the stem and bulb nematode, Ditylenchus dipsaci, from Eastern Canada By Sandra Poirier Student ID: 260751029 Department of Plant Science McGill University Montreal Quebec, Canada October 2018 A thesis submitted to McGill University in partial fulfillment of the requirements of the Degree of Master in Plant Science © Sandra Poirier 2018 Table of content List of figures ...................................................................................................................... 4 List of tables ........................................................................................................................ 5 Abstract ............................................................................................................................... 6 Résumé ................................................................................................................................ 8 Acknowledgements ........................................................................................................... 10 Contribution of Authors .................................................................................................... 11 Chapter 1: Introduction ..................................................................................................... 12 Chapter 2: Literature review ............................................................................................. 14 2.1. Ditylenchus dipsaci ............................................................................................ 14 2.1.1. Biology ........................................................................................................ 14 2.1.2. Life cycle .................................................................................................... 16 2.1.3. Symptoms & damages ................................................................................ 17 2.1.4. Propagation ................................................................................................. 18 2.1.5. Host plants .................................................................................................. 18 2.1.2. Management ................................................................................................ 21 2.1.7. Extraction methods ..................................................................................... 23 2.1.8. Population Genetics .................................................................................... 27 Chapter 3: Hypothesis and Objectives .............................................................................. 30 Chapter 4: Validation of extraction methods used for the diagnostic of the stem and bulb nematode, Ditylenchus dipsaci ......................................................................................... 31 4.1. Abstract .................................................................................................................. 31 4.2. Introduction ............................................................................................................ 32 4.3. Material & Methods ............................................................................................... 33 4.4. Results .................................................................................................................... 37 4.5. Discussion .............................................................................................................. 38 4.6. Acknowledgement .................................................................................................. 40 Connecting statement ........................................................................................................ 41 Chapter 5: Host range and genetic characterization of Ditylenchus dipsaci populations from Eastern Canada ......................................................................................................... 42 5.1. Abstract .................................................................................................................. 42 5.2. Introduction ............................................................................................................ 43 5.3. Materials and methods ........................................................................................... 46 5.4. Results .................................................................................................................... 52 5.5. Discussion .............................................................................................................. 55 5.6. Acknowledgments .................................................................................................. 58 Chapter 6: Discussion ....................................................................................................... 61 Chapter 7: Conclusions and contribution to knowledge ................................................... 65 References ......................................................................................................................... 67 List of figures Figure 1: Diagram of Ditylenchus dipsaci larvae ............................................................. 14 Figure 3 : Diagram of the density flotation method with a sugar solution. (Poirier S. 2018) ................................................................................................................................. 24 Figure 4: Schematically representation of Oostenbrink elutriator (MEKU 2017). .......... 25 Figure 5 : Diagram of a Baermann's funnel assembly: ..................................................... 26 Figure 6: Diagram of the Baermann-pan assembly (Poirier S. 2017) ............................... 27 Figure 7: Efficacy of three different methods for the extraction of Ditylenchus dipsaci from four soil types expressed as the percentage of recovery. C= silty clay; L=loam; M=muck; S=sand. ............................................................................................................. 37 Figure 8: Host range of 4 populations of Ditylenchus dipsaci from Quebec and Ontario.53 Figure 9: Principal component analysis of 32 populations of Ditylenchus dipsaci based on genome-wide allele frequencies of 481 loci with no missing data and a minimum coverage of 20 reads/locus/population. ............................................................................ 54 List of tables Table 1. Number of nematodes extracted from garlic bulbs or from stems and leaves using three different extraction methods. .......................................................................... 38 Table 2. Primers used in the PCR reaction to identify Ditylenchus species ..................... 46 Table 3. Samples of Ditylenchus dipsaci used for genetic characterization. .................... 50 Supplementary Table 1. Fixation index (Fst) values for 32 populations of Ditylenchus dipsaci based on genome-wide allele frequencies of 481 loci with no missing data and a minimum coverage of 20 reads/locus/population. ............................................................ 60 Abstract The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode responsible for major economic loss worldwide. A wide host range, reaching more than 500 plants species and the presence of more than 30 biological races defined by their host preference, make this species one of the most difficult nematodes to manage. Since 2011, garlic producers from Ontario and Quebec have been particularly affected with economic losses caused by this pest. An appropriate extraction method is essential for the detection of the nematode. However, the extraction efficacy will be influenced by many factors such as the nematode species (size and motility), the material to be diagnosed (soil or plants tissues) and the type of soil or plants tissues. Therefore, many different methods exist, each with their own advantages and disadvantages. Three extraction methods (Baermann pan, Baermann funnel and sonication) were compared for the extraction of nematodes from garlic bulbs, stems and leaves. Three methods (Baermann pan, Baermann funnel and sugar flotation) were also tested on four types of soil sample. This study confirmed the efficiency of the Baermann pan with D. dipsaci, both with soil and plant tissue samples. Reproduction of D. dipsaci on a particular host depends on its biological race, which was unknown for the populations of eastern Canada. In this study, the host range of four populations of D. dipsaci from Quebec and Ontario was determined in a greenhouse test on 11 crops. Garlic, onion and green onion showed great susceptibility to the nematode while reproduction on potato was poor. No development was observed on corn, soybean, barley, alfalfa, mustard, carrot, and lettuce. These plants could therefore be used in rotation crops in a control program. Finally, thirty-two populations of D. dipsaci were genetically characterized using genotyping-by-sequencing. Comparison of allele frequencies at 481 SNPs showed that most of the populations had a genotype similar to a reference population from northern Ontario. However, a sample from eastern Quebec exhibited a very distinct genotype and will require further phenotyping in the greenhouse to preclude the possibility of a different race. Résumé Le nématode des tiges et des bulbes, Ditylenchus dipsaci, est un nématode phytoparasite responsable de pertes économiques majeures dans le monde entier. Une vaste gamme d'hôtes, atteignant plus de 500 espèces végétales