Host range and genetic characterization of the stem and , 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. ...... 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 extracted from or from stems and leaves using three different extraction methods...... 38 Table 2. Primers used in the PCR reaction to identify Ditylenchus ...... 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, and green onion showed great susceptibility to the nematode while reproduction on 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 et la présence de plus de 30 races biologiques définie par la préférence de l'hôte font de Ditylenchus dipsaci l'une des espèces de nématodes phytoparasites les plus difficile à gérer. Depuis 2011, les producteurs d'ail de l'Ontario et du Québec ont été sévèrement touchés par ce problème.

Une méthode d'extraction appropriée est essentielle à la détection du nématode dans un

échantillon. L’efficacité de l’extraction dépend de nombreux facteurs tels que l'espèce de nématode (taille et mobilité), le matériel à diagnostiquer (sol ou tissus) et le type de sol ou de plante. De nombreuses méthodes sont disponibles avec leurs avantages et leurs inconvénients. Trois méthodes d'extraction (Assiette de Baermann, entonnoir de

Baermann et sonication) ont été comparées pour l'extraction des nématodes dans les bulbes, les tiges et les feuilles d'ail. De plus, trois méthodes (Assiette de Baermann, entonnoir Baermann et flottaison au sucre) ont été comparées pour l'extraction des nématodes dans quatre types de sol. Cette étude a confirmé l'efficacité de l’assiette de

Baermann avec D. dipsaci, à la fois avec des échantillons de sol que des tissus végétaux.

La reproduction de D. dipsaci sur un hôte particulier dépend de sa race biologique. Dans cette étude, le spectre d'hôte de quatre populations de D. dipsaci du Québec et de l'Ontario a été déterminé à l'aide d'un essai en serre sur 11 cultures. L'ail, l'oignon et l'oignon vert ont montré une grande sensibilité au nématode alors que la reproduction sur pomme de terre était faible. Aucun développement n'a été observé sur le maïs, le soja,

l'orge, la luzerne, la moutarde, la carotte, et la laitue. Ces plantes pourraient donc être utilisées comme cultures de rotation dans un programme de lutte.

Finalement, trente-deux populations de D. dipsaci ont été caractérisées génétiquement en utilisant le génotypage par séquençage. La comparaison des fréquences alléliques de 481 polymorphismes nucléotidiques simples (SNP) a montré que la plupart des populations du Québec et de l'Ontario avaient un génotype similaire. Par contre, une population du

Québec a démontré un génotype très différent et exigera des phénotypages supplémentaires afin de confirmer s’il s’agit d’une race différente.

Acknowledgements

Many persons contributed to the success of this project and I would like to show them all my gratitude.

Among them, I would firstly thank my Co-supervisor Dr. Benjamin Mimee. Thank you for giving me the opportunity to get out of my comfort zone by doing this project in this new discipline for me. Thank for your guidance and great advices all along this project.

Thank to my supervisor Dr. Valérie Gravel for all you precious advices. You always took time to answer all my questions and correct my work and I am very grateful.

Thank to Pierre-Yves Véronneau for all the help and explanations with the bioinformatics and molecular stuff, you've been an excellent tutor and I would never had done it without you.

Thank to Nathalie Dauphinais for sharing your passion with me and your devotion for this field of study. Nothing would have been possible without your precious knowledge, your patience and your emotional support.

Finally, thanks to all my labmates who helped me with my manipulations and contributed to make those years an unforgettable experience.

Contribution of Authors

This document has been written using the manuscript-based thesis format following the

“Guidelines for Thesis Preparation and Submission” as required by the Department of

Plant Science and Graduate studies at McGill University. The project was done under the supervision and co-authorship of Dr. Valérie Gravel and the co-supervision and co- authorship of Dr. Benjamin Mimee with the financial support of Agriculture and Agri- food Canada.

The candidate has conducted all the assays for the host range evaluation in greenhouses, the comparison of the extraction methods and the genetic characterization of the stem and bulb nematodes. The author, Sandra Poirier, also performed all the data analysis and wrote the two manuscripts included in this thesis.

Dr Benjamin Mimee and Dr. Valérie Gravel have provided the funding and technical support for the research, designed the experiments and contributed to data analysis and critical review of the manuscripts.

Mr Guy Bélair and Ms Nathalie Dauphinais contributed to the experimental design, data analysis and review of the two manuscripts.

Mr Hervé Van Der Heyden has provided biological material and contributed to the manuscript on host range evaluation.

Chapter 1: Introduction

The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode affecting over 500 plant species worldwide (Sturhan & Brzeski 1991). It is generally found in temperate regions but its presence has also been recorded in extreme temperature areas (Viglierchio 1971). D. dipsaci is listed as a quarantine pest by the

European and Mediterranean Plant Protection Organization (EPPO 2004). In Canada, it is subjected to many directives by the Canadian Food Inspection Agency (CFIA) and is considered as a quarantine pest on potatoes (CFIA 2013). These regulatory differences complicate access to the export market for Canadian producers.

The nematode’s presence on onion and garlic was known in many provinces in

Canada for a long time (Mountain 1957; Johnson & Kayler 1972; Fushtey & Kelly 1975).

Since the 2000's, garlic production gained in popularity in the provinces of Ontario and

Quebec (Statistic Canada 2016). With the increase of this crop, large outbreaks of D. dipsaci were observed recently in garlic fields in Ontario and Quebec (RAP 2011).

Unfortunately, these infestations resulted in important crop losses of up to 90 % (Abawi

& Moktan 2010; Celetti 2011).

Early and effective detection of D. dipsaci is necessary for the implementation of good management practices. However, the classic soil extraction procedures for nematodes are thought to have a limited efficiency with this large nematode (Bélair

2016). Thus, comparison and validation of extraction methods could provide useful guidelines for diagnostic laboratories.

Management practices are also limited. Using nematode-free seeds and avoiding infested sites (Abawi & Moktan 2010) are the two most important methods.

Unfortunately, nematode-free certified garlic seeds are not currently available in Canada.

Some cleaning treatments for infected seeds could be done like hot water treatment or the use of chemical compounds. Unfortunately, those treatments are not always effective

(Krusberg 1961), may reduce seed germination and are hazardous for human (Kim & al.

2017) and environmental health (Solomon & Ravishankara 1992). Furthermore, the cultivation of garlic in Quebec is mainly under organic production. Crop rotation with non-host plants is a management strategy that would be much safer and sustainable.

Finding non-host plants is therefore a priority for the management of D. dipsaci in

Québec and Ontario.

Establishing the host range for every population of D. dipsaci on every crop would be extremely time consuming. Thus, the genetic characterization of several populations of this pest could help to evaluate if they are related or not and to compare their host range in relation to their genetic background.

So far, very little is known about the population of D. dipsaci found in Eastern

Canada in garlic fields. The objectives of this project were to find the most appropriate extraction methods for the stem and bulb nematode; to establish its host range and to characterize the genetic diversity of populations from Quebec and Ontario.

Chapter 2: Literature review

2.1. Ditylenchus dipsaci

2.1.1. Biology Ditylenchus dipsaci (Kühn 1857) Filipjev is an endoparasitic nematode (Duncan & al. 2006) of the Anguinidea family. No longer than 1.5 mm, it has a long slender shape, a pointed tail (Abawi & Moktan 2010) and a short stylet measuring 10-12 µm (Brzeski

1991). The species is not presenting a lot of morphological differences with the other members of the genus Ditylenchus (Wendt & al. 1993) with the exception of the four incisions on the lateral field (Hooper 1972) (Figure 1). Morphological characters are variable between development stages (Brzeski 1991), making identification difficult.

Figure 1: Diagram of Ditylenchus dipsaci larvae (Hooper, 1972)

As suggested by its name, the stem and bulb nematode is found mainly in bulbs and stems, but also in leaves and seeds and to a lesser extent, in soil (Hooper 1972). However, it is rarely found in roots (Decker 1989). In contrast with sedentary nematodes that induce a permanent feeding site, this migratory parasite feeds and reproduces in the parenchymatous tissues while migrating in plant cells (Duncan & al. 2006; Hooper

1972). The nematode secretes pectinases that dissolve the lamellae between cells

(Krusberg 1961) causing the breakdown of the cellular integrity and the tissue destruction.

The species D. dipsaci is distributed worldwide (CABI 2015). It is well adapted to temperate regions (Sturhan & Brzeski 1991) and its presence has been recorded from hot desert to cold mountain climates (Viglierchio 1971). When temperature and humidity conditions become unfavorable, numerous larvae of D. dipsaci will clump together in a mass described as "eelworm wool" (Ellenby 1968). Then, the fourth larval stage of D. dipsaci will desiccate and enter in a state of anhydrobiosis. In this metabolic stage, some laboratory specimens were found to survive for 23 years (Fielding 1951). In field conditions, the survival of D. dipsaci in debris or directly in soil when no host is available was found to be four years (Sturhan & Brzeski 1991).

In the family, Ditylenchus dipsaci is the species that has the greatest economic impact (Sturhan & Brzeski 1991). For that reason, it was listed among the top five plant-parasitic nematodes of importance worldwide (Jones & al., 2013). The presence of D. dipsaci in Canada has been known for a long time, locally affecting onion

(Mountain 1957, Johnson & Kayler 1972) and garlic (Fushtey & Kelly 1975) but large outbreaks are much more recent. In 2011, a wide survey in Ontario that included more

than 100 fields, revealed that 73 % of the garlic samples were infested by D. dipsaci

(OMAFRA 2011). In the province of Quebec, the stem and bulb nematode was not considered problematic until 2010 (RAP 2013). In 2012, the Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (MAPAQ) did a small survey on 15 garlic samples in Quebec; eight of them were infested (RAP 2013). Some information suggests that the infestation in Quebec could be the result of the introduction of an infested seed lot from Ontario.

2.1.2. Life cycle

The life cycle of D. dipsaci begins after the egg deposition (Figure 2). The female produces from 200 to 500 eggs (Yuksel 1960). Second-stage larvae (J2) will hatch from the egg after 7 days at 15°C (Yuksel 1960). The J2 larvae use their stylet to pierce the eggshell and their head to break it (Perry & Moens 2011). After hatching, all life stages of D. dipsaci have the ability to infect plant tissues. The fourth stage larvae, with their desiccation capacities, are considered as the main infectious stage (Sturhan & Brzeski

1991) du to the survival capacity. The species is amphimictic, meaning that both genders are required to produce an offspring (Perry & Moens 2011). The life cycle is completed after 19 to 23 days in onion seedlings at 15 °C (Yuksel 1960). The longevity of D. dipsaci is very variable due to its capacity of desiccation and will depend on soil

conditions and the availability of a susceptible host.

Figure 2: Diagram of the life cycle of Ditylenchus dipsaci in plant tissues. (Agrios 2005) 2.1.3. Symptoms & damages

Most of the damages causes to the plant cells by D. dipsaci are the direct results of feeding and migration (Yeates & al. 1993). By secreting digestive enzyme, the nematodes dissolve the cell middle lamellae (Duncan & al. 2006). The resulting symptoms are generally unspecific, which complicates diagnosis and the management. In the early stage of the infection, garlic plants can be asymptomatic (Celetti & Hughes

2011). When severely infected, garlic leaves becomes yellow and collapse (Abawi &

Moktan 2010) while the stem become distorted and stunted (Hooper 1972). The root plate will eventually separate from the bulb (Celetti 2009) and the plants may die prematurely

(Hooper 1972). The bulbs will show discoloration, rotting on the base (Diekmann 1997),

and will appear shrunken and be lighter in weight (Abawi & Moktan 2010). Cloves can turn yellow and separate from each other (Diekmann 1997). Those damages also increase the risk of secondary infections like Fusarium basal plate rot, caused by Fusarium oxysporum f.sp. cepae, a well-known pathogen in garlic fields (Celetti 2016). Onion plants, when infected, will also present twisting, swelling and discoloration of leaves and stems (Abawi & Moktan 2010). The presence of D. dipsaci cause important crop losses reaching 60-80% in onion and garlic (Sturhan & Brzeski 1991). In Ontario, loss of 100% has been recorded (Celetti 2011).

2.1.4. Propagation

D. dipsaci, with its desiccation capacity, can survive in soil, in the dry plant debris and in stored plant materials (Decker 1989). The use of infested seeds and cloves is the first cause of contamination (Abawi & Moktan 2010) spreading the pest between different fields. The presence of larvae in plant debris or in the soil also contribute to the spread of the pest. This nematode is able to move into a small film of water. Irrigation systems and heavy rains are therefore other vectors of propagation from plant to plant within a field (Decker 1989).

2.1.5. Host plants

Racial characterization is an important challenge for D. dipsaci (Viglierchio 1971). The species has a very large host range estimated at over 500 plant species and is separated in more than 30 different biological races (Sturhan & Brzeski 1991) based on their host

preferences. Those having the greatest economic importance include the sugar beet, onion, , oat, strawberry, alfalfa, and tulip races (Sturhan & Brzeski 1991). Most of these races of D. dipsaci are polyphagous and will develop on a wide range of plants

(Viglierchio 1971). Literature reports many cases where D. dipsaci found in garlic also reproduces on onion ( cepa) (Douda 2005), and on other Allium but also on unrelated plants including snap bean and (Edwards & Taylor 1963). Some plants can also be good hosts for several races of D. dipsaci (Janssen 1994).

Distinction of these races using morphological observations has proven to be unsuccessful (Barraclough & Blackith 1962). Hybridization assay with many races of D. dipsaci demonstrated that the definition of the race concept is not accurate (Eriksson

1974).

2.1.5.1. Garlic

The recent large infestation of D. dipsaci in Canada was reported on garlic.

Garlic, Allium sativum, is a plant from the Allium genus which includes onion, leek and shallot (Diekmann 1997). In 2013, the worldwide garlic production was 24,255,302 metric tons (FAOSTAT 2013). In 2015, 368 metric tons were produced in Canada

(CANSIM 2016). Garlic is a perennial plant. A cold temperature of 0 to 5°C is necessary to initiate growth and for bulb formation (Mann & Minges 1958). Cloves obtained by a previous culture or a provider are planted in fall (half September to the end of October)

(OMAFRA 2009) or the early spring (April to May). The harvest is done in July. Garlic is grown in all soil types, including muck soil. As large proportion of the garlic production in Canada is often on small acreage and under organic management, chemical control of pests and diseases is not an option.

Allium sativum species is divided in two subspecies, softneck (sativum) and hardneck (ophioscorodon) (Block 2010). Although both types are cultivated in eastern

Canada, the hardneck subspecies is more adapted to northerly climate (Block 2010).

More than eight varieties of the subspecies Allium sativum var. ophioscorodon are currently cultivated in eastern Canada but the group porcelain, with the well-known variety Music, is the more common one (Ail Québec 2017, OMAFRA 2009). Allium sativum var. sativum has two varieties cultivated in Quebec, Silverskin and Artichoke

(Ail Québec 2017).

2.1.5.2. Onion

The onion (Allium cepa), is a biennial, herbaceous plant of the Amaryllidaceae family. It is Canada's most important condiment crop (Nuttall 2013). Production of dry onion in Quebec and Ontario represents a farm gate value of 67 million dollars (Statistics

Canada 2016). In Eastern Canada, dry yellow or red onion, as well as Spanish , green onions (bunched) and French shallots are cultivated (PRISME 2016). Yellow and red onions are usually produced from the seed, while French shallot is usually grown from clove. Spanish onion are produced from glasshouse transplants planted at the begging of the summer (PRISME 2016). The growth time is relatively between 90 and

115 days. Seeding or planting in Canada begins on mid-May and the harvest is in the end of August.

2.1.5.3. Other crops

The host preferences of Canadian populations of D. dipsaci have been evaluated on common pulse crops (yellow , chickpea, common bean and ), spring wheat,

canola, creeping thistle and garlic (Hajihassani & al.2016). The results of this study showed that D. dipsaci populations from gralic reproduced on pea varieties, chickpea and bean.

Infested garlic grown in mineral soils was often found close to major field crops like soybean, corn and cereals. Soybean is one of the principal field crops in Canada

(Statistics Canada 2015). Only in the province of Quebec, one million tons have been produced in 2015 (Statistics Canada 2015). Unfortunately, soybean has been reported as a host for a D. dipsaci population isolated from onion (Edwards & Taylor 1963). This increases the need of more knowledge on the effect of D. dipsaci population in Eastern

Canada to prevent important economic losses. A host range study with plant species currently grown in Canada was therefore necessary for risk assessment and an effective use of crop rotation. Currently, the D. dipsaci outbreak in Quebec seems to be limited to garlic.

2.1.2. Management

The first management method is to avoid the introduction of infested materials and to use only nematode-free seeds (Abawi & Moktan 2010). Unfortunately, even asymptomatic bulbs can be highly infected (Celetti & Hughes 2011), and there is no nematode-free seed certification program in Canada yet (Greig 2017). Garlic Growers

Association of Ontario (GGAO) were working on the production of clean garlic seed

(Celetti & Hughes 2011) from bulbils in greenhouse but the program has been suspended.

Treating garlic bulbs in a hot water bath is another method that is used to obtain clean cloves. Cloves have to be incubated in 38 to 49 °C water for one to three hours

depending of the water temperature (Hooper 1972, Diekmann 1997, Abawi & Moktan

2010). However, this technique is tricky as overheating (over 50°C) will damage the bulbs and prevent germination (Cantwell & al. 2003) and too low temperatures will not kill the nematodes. Soaking the seeds in nematicidal or fumigation products has also been proposed but it would expose agricultural workers to noxious chemicals (Roberts &

Matthews 1995).

Uninfected seeds have to be planted into a nematode-free site. Soil fumigation with methyl bromide was an effective control method for D. dipsaci (Siti & al. 1982) but this product has important ozone depletion potential (Solomon & Ravishankara 1992) and is toxic for humans (Calvert & al. 1998). Methyl bromide is prohibited (with exceptions) in Canada since 2005 (Government of Canada 2004). Fumigation is still a recommended method to clean the soil (Abawi & Moktan 2010) but, in Canada, there is no product currently approved for D. dipsaci. Biofumigation trials with mustard have been done but even if populations of D. dipsaci in soil decreased, it was not as effective as fumigation (Celetti 2009).

The last, and probably the oldest method of control is crop rotation. Four-year rotations without host plant is a good and simple way to limit damages caused by D. dipsaci (Sturhan & Brzeski 1991, Celetti 2009). However, the host range of D. dipsaci varies greatly between populations. Thus, using this method requires a good knowledge of the populations in the field and the plants they can infest. Crop rotation combined with the used the biofumigation could be an effective way to control the presence of D. dipsaci without pesticides.

2.1.7. Extraction methods

The determination of nematodes presence requires adapted extraction methods. As mentioned by the European and Mediterranean Plant Protection Organisation, there is no available method that is effective for all nematode species and under all conditions

(EPPO 2013). Extraction methods are based on three mains principles: the specific density of nematodes, their size and shape and their motility (Hooper & al. 2005). The stem and bulb nematode, as a migratory nematode, can be found in plant material and in the soil.

The sugar flotation (Fig. 3) is a popular method to extract nematodes from soil samples. It is based on the difference of densities between soil particles and nematodes

(Viglierchio & Yamashita 1983) and allows for the recovery of all larval stages, whether dead or alive (Van Bezooijen 2006). The soil sample is usually mixed with tap water in order to dissolve some soil particles and remove soil heaps. The solution is centrifugated a first time to remove light particles and debris. The pellet is then dissolved into a solution with a specific higher density like solutions of sugar, MgSO4 or ZnSO4 (Van

Bezooijen 2006) and centrifugated again. The nematodes remain in the supernatant while soil particules sink. Although the method recovered all larval stages dead or alive, it required expensive equipment. Furthermore, the extraction solution can be toxic for the nematode or, by increasing osmotic tension, damaged more nematodes (Van Bezooijen

2006).

Figure 3: Diagram of the density flotation method with a sugar solution. (Poirier S. 2018)

Another extraction method for soil is elutriation. The Oostenbrink elutriator (Fig.

4) is based on the separation of different particles by their density, shape and size using a controled water flow (Van Bezooijen 2006). Soil samples are disolved into a large amont of water. A controled upper currrent of water separates the particles in the sample.

Debrits with higher density sink while the particules with a specific density will be sieved and recovered for nematode enumeration. Dead and alive larvae can be separated from soil particles. However, this methode required expensive equipment and is time consuming since only small samples can be treated.

Figure 4: Schematic representation of Oostenbrink elutriator (MEKU 2017).

The Baermann’s funnel (Baermann 1917) is an extraction technique used for both soil and plant material. A funnel ended with a plastic hose is hung on a support (Figure

5). The sample is put in a filter, placed on a sieve at the top of the funnel which is then filled with tap water. Nematodes will swim to the bottom of the funnel. Based on the mobility of the nematode, it requires simple and cheap installation but has several drawbacks. The major ones are the lack of oxygen at the end of the funnel that can kill the nematodes and the impossibility to recover dead or dormant nematodes as well as resting structures like cysts or nemawool. Nevertheless, this is the recommended method by EPPO for D. dipsaci (EPPO 2008).

Figure 5 : Diagram of a Baermann's funnel assembly: F, glass or plastic funnel; W, tap water ; FL (dashed line) fluid level in funnel; SB, Sample basket shown to right in exploded view (FCC, samples; KW, two layers of Bounty LRM Lucite ring with nylon mesh affixed with cement; T, rubber or plastic tubing; PC, pinch clamp; CV, catch vessels, conical centrifuge tube, or beaker. (Lok 2007)

The Baermann’s pan (B-pan) is a modified version of the Baermann’s funnel, using a pan instead of a funnel (Figure 6) (Forge & Kimpinski 2006). Used for soil and plant material, this technique allows a better oxygenation of the water. The B-pan could be a good alternative to the recommended Baermann’s funnel but still won’t catch dead/dormant nematodes. Even with alive and mobile nematodes, these two methods are reported to have a low recovery rate of around 30%.

Figure 6: Diagram of the Baermann-pan assembly (Poirier S. 2017)

Recently, a new method using ultrasounds has been proposed for nematodes extraction from plant tissues (Tangchitsomkid & al. 2015). Adapted for migratory nematodes, this method is relatively simple and fast. Plants tissues are placed in tap water and then, exposed to ultrasounds at a frequency of 40 KHz for 40 minutes. This method has not been tested with D. dipsaci but showed good results with other plant-parasites like Meloidogyne incognita, Helicotylenchus dihystera, Pratylenchus penetrans,

Ditylenchus triformis, and Tylenchorhychus sp (Tangchitsomkid & al. 2015)

Even if those extraction methods have been recommended by the EPPO for D. dipsaci (EPPO 2013) (with the exception of the ultrasonic method), data of recovery with garlic populations of the stem and bulb nematode are almost non-existent.

2.1.8. Population Genetics

The host range variation between each biological races of D. dipsaci make identification and recommendation to growers difficult. Thus, the need for good molecular identification tools is very high. The use of non-host plant for crop rotation is a good method of management, but it requires prior phenotype and genetic knowledge of

the populations present in the field. The use of molecular tools is helpful to distinguish important genetic polymorphisms and differentiate populations (Esquibet & al. 2003).

For now, there are no quick and reliable molecular tools to differentiate the races of D. dipsaci. In the last decade, molecular tools have been developed for the differentiation of

D. dipsaci from similar species, including the quarantine species D. destructor, found in

Ontario (Yu & al. 2012) and Ditylenchus weischneri (Madani & al. 2015), a species reproducing on Canada thistles in Manitoba. A multiplex PCR assay has also been developed for the molecular identification of Ditylenchus dipsaci, and Ditylenchus gigas (Jeszke 2015)

The analysis of the genetic variation among the D. dipsaci populations in Ontario has distinguished two genetic isolates (Qiao & al. 2013). The genetic comparison of isolates from different provinces and countries will provide new information on risk assessment for this species. Eventually, whole genome comparison of many isolates could also allow the development of genetic markers for each population phenotypes.

There is no complete genome sequence of D. dipsaci available yet but a draft genome has been published (Yu & al. 2014). Sequencing the whole genome of an organism is still expensive and cannot be done on a large number of samples. However, some techniques allow a rapid comparison of a subset of the genome. Genotyping by sequencing (GBS) is a powerful tool to rapidly identify genetic markers like single nucleotide polymorphism

(SNP) between populations. An approach for high diversity species has been developed recently (Elshire & al. 2011). Restriction enzymes (RE’s) are used to reduce the genome complexity. Then, barcode adaptors are ligated to the DNA fragment. Samples of DNA are pooled, cleaned and a PCR is run. Once the library preparation is done, samples are

sequenced using next-generation sequencing. An adaptation of this method has been successfully used with the golden cyst nematode Globodera rostochiensis (Mimee & al.

2015).

Chapter 3: Hypothesis and Objectives

For the first hypothesis, we think that the Baermann funnel is unadapted for the detection of D. dipsaci in soil sample because of its low efficacy for this large nematode.

Elutriation will probably be more efficient due to its combination of multiple separation principles and the recovery of both alive and dead larvae (Van Bezooijen 2006). It is also predicted that the ultrasonic extraction will be more efficient for the recovery of nematode in bulbs and stems.

The first objective of this thesis is to validate the extraction methods (Baermann funnel, Baermann pan, elutriation and sugar flotation) currently used for the diagnostic of

D. dipsaci.

The second hypothesis is that no difference of host range is expected between populations from Ontario and Quebec. Also, according to the literature, nematode development should be important on garlic, onion, and carrot.

The second objective is to determine the host range of four populations of D. dipsaci from Quebec and Ontario.

For the last hypothesis, it is suggested that because of their common origins, genotypic similitudes should be observed between the populations from Quebec and

Ontario while differences with international populations are expected.

The last objective is to genetically characterize D. dipsaci populations from

Europe, Ontario and Quebec using genotyping-by-sequencing.

Chapter 4: Validation of extraction methods used for the diagnostic of the stem and bulb nematode, Ditylenchus dipsaci

4.1. Abstract

The stem and bulb nematode, Ditylenchus dipsaci (Kühn) Filipjev, is a serious threat to many important crops worldwide, including garlic. For this crop, effective detection methods are essential to discard infected seed cloves and avoid contaminated fields. This study compared the efficacy of different extraction methods for soil and garlic tissues infested with this nematode. The Baermann pan, Baermann funnel and sugar flotation were compared on four types of soil previously inoculated with D. dipsaci. Two Baermann methods were also compared to a sonication technique for the extraction of nematodes from garlic stems, leaves and bulbs. This study confirmed the validity of the Baermann pan and funnel methods for the extraction of D. dipsaci from soil and garlic tissues. The sugar flotation and sonication procedures yielded significantly fewer nematodes and were not considered adapted for the current purpose.

4.2. Introduction

The stem and bulb nematode, Ditylenchus dipsaci (Kühn) Filipjev, is known to cause severe damages to a wide range of crops (Sturhan & Brzeski 1991). Garlic and onions are particularly affected and yield losses reaching 90% are reported (Celetti 2011).

Strict measures should be taken to avoid the introduction and contamination of the stem and bulb nematodes in new fields. Good management practices start by the use of nematode-free seeds and avoiding already infested fields (Abawi & Moktan 2010).

Therefore, precise and sensitive extraction methods are needed. Because D. dipsaci can be retrieved from soil but mainly in the stems and the bulbs, the perfect extraction methods should accommodate all of these materials. Also, onions and especially garlic are grown in very diverse soil types including organic (muck) soil and different types of loam. Thus, the extraction method should not be influenced by soil parameters.

Unfortunately, the European and Mediterranean Plant Protection Organisation (EPPO) confirmed that there is no available method that is effective for all nematode species under all conditions (EPPO 2013). Some compromises are therefore usually made and the actual extraction efficacy is not always known.

Extraction methods are based on three main principles: specific density of the nematodes, size and shape of the nematode and its motility (Van Bezooijen 2006). Many methods are proposed for the extraction of nematodes from soil samples but their efficacies are influenced by many factors such as the nematode species and soil type

(Viglierchio 1983). The Baermann funnel and Baermann pan are used for soil and plant samples. These two methods rely on the motility of the nematode to leave the soil or plant tissue (Van Bezooijen 2006). They are inexpensive and simple to perform and their

recovery rate is estimated to be between 50% and 80% (EPPO 2013). These methods are recommended by the EPPO (EPPO 2013) and are currently used by many diagnostic laboratories. Another method recommended for soil samples is the centrifugal flotation method. This technique allows the recovery of both active and inactive nematodes using the difference in specific gravity between nematodes and soil particles (Van Bezooijen

2006). This method is more expensive but was shown to have a higher extraction efficiency on various species (Van Bezooijen 2006). A new method proposed by

Tangchitsomkid et al. (2015) use ultrasounds to recover nematodes from plant tissues.

This method is simple and time effective but requires expensive material. It has been tested with other migratory parasitesbut not with D. dipsaci. All these methods are recommended for different species of nematodes but very few reports have confirmed their utility for D. dipsaci. Therefore, the efficacy of the Baermann pan, Baermann funnel and sugar flotation were evaluated for the recovery of known numbers of stem and bulb nematodes from four soil types. Also, we compared the Baermann pan and funnel methods to a sonication method for the extraction of D. dipsaci from garlic bulbs, stems and leaves.

4.3. Material & Methods

Stem and bulb nematode culture. A population of D. dipsaci originating from

Québec, Canada was isolated from garlic bulbs with the Baermann funnel method and reared in Petri dishes on pea sprouts (pea cv. Green Arrow) growing on Gamborg B-5 medium with minimal organics (3.2 g/L; Sigma-Aldrich, CA) containing agar (8g/L;

Anachemia, CA) and sucrose (20 g/L; BioShop®, CA, USA). Petri dishes were incubated

in the dark at 23°C during three months. Nematodes were then extracted by placing the

Gamborg B-5 medium with the pea sprout in a disposable wiper (Kimwipes®, Kimberly-

Clark, CA) in another Petri dish filled with tap water. Nematodes were recovered two hours later in the water, counted under a stereomicroscope using a counting slide to adjust the concentration at 200 larvae/cc of water and used immediately to spike soil samples.

Soil samples and inoculation. Four pasteurized soil types were tested: sand (97% sand, 1% silt and 2% clay), loam (42% sand, 34% silt and 24% clay), silty clay (14% sand, 39% silt and 47% clay), and muck soil. Volumes of 100-cc of each soil type were measured and transferred into 150-ml plastic containers. Before inoculation with nematodes, tap water was added to homogenize soil moisture (loam = 25 ml; clay = 20 ml; muck = 15 ml; and sand = 10 ml). Each soil sample was inoculated with exactly 200 nematodes (mixed stages) contained in one cc of water. The inoculum was poured into a hole (1-cm diameter, 2-cm depth) into each soil sample and incubated at room temperature for 2 hours. Ten replicates of each soil type were done for each of the three soil extraction methods tested (Baermann funnel, Baermann pan and centrifugal sugar flotation).

Garlic samples. Garlic plants naturally infested with the stem and bulb nematode were harvested at the experimental farm of Agriculture and Agri-Food Canada in Saint-

Clothide, Québec. All bulbs were cut in small pieces, mixed together and separated in three homogenous pools. Stems and leaves were also cut in small sections, mixed together and split in three homogenous parts. Nematodes were then extracted from these pools using one of the following methods: Baermann funnel, Baermann pan and

Ultrasonic extraction. Ten replicates were done for each technique by randomly sampling

30 g of bulb or 13 g of stem and leave material from each pool.

Baermann funnel method. This method was initially developed by Baermann

(1917) and modified by Whitehead and Hemming (1965). Spiked soil (100 cc), infested bulb or stem and leave material (30g and 13g, respectively) were placed on two sheets of paper towel (Bounty©, PROCTER & GAMBLE, USA) previously placed on a mesh inside a funnel ending by a clamped rubber hose. Tap water was added half way up the mesh. After seven days at room temperature for soil samples and three days for plant tissue samples, nematodes were recovered in the water at the bottom of the tube and poured through a 25-µm sieve. Nematodes were then counted under a stereomicroscope using a counting slide.

Baermann pan method. This modification of the Baermann funnel was proposed by Oostenbrink (1954) and uses a dish instead of a funnel. Spiked soil (100 cc), infested bulb or stem and leave material (30g and 13g, respectively) were placed on two sheets of paper towel (Bounty©) previously placed on a 15-cm round mesh placed in a 16-cm round plastic plate (plant pot saucer). One hundred ml of tap water were added half way up the mesh. After seven days for soil samples and three days for plant tissue samples at room temperature, nematodes were recovered from the water at the bottom of the pan by pouring it through a 25-µm sieve. Nematodes were then counted under a stereomicroscope as described previously.

Centrifugal sugar flotation. This method was proposed by Caveness and Jensen

(1955). One hundred cc of spiked soil samples were suspended in 1 L of tap water.

Samples were then sieved through a 200-µm sieve and after it was rinsed, soil material

and debris were discarded. Samples were then sieved again through a 25-µm sieve, well rinsed with tap water and poured into a 50-ml tube. Tubes were centrifuged at 1800 g for four minutes and supernatant was transferred into a new tube for further rinsing and sieving. Pellets were resuspended in a sugar solution (484 g/l) (Van Bezooijen 2006) to a volume of 40 ml and vortexed. Then, tubes were centrifuged at 1800 g for one minute and supernatant was pooled with the previous one. Pooled supernatants were poured through a 25-µm sieve and rinsed with tap water to remove the sugar solution.

Nematodes were collected from the sieve and count under a stereomicroscope as described previously.

Ultrasonic extraction. Plant tissue samples (30 g of bulb or 13 g of stem and leave material) were placed into a 500-ml beaker filled to the top with tap water and placed in a Tabletop Ultrasonic Cleaners (FS220H; Fisher Scientific, USA) at 40 kHz for

40 minutes. The suspension was poured through a 25-µm sieve and nematodes were counted with a stereomicroscope as described previously.

Statistical analyses. Statistical analyses were performed with Rstudio (R3.3.2,

RStudio, Inc., USA). Normality of distribution of the data was evaluated with the

Shapiro-Wilk test. As the quantitative descriptor did not obey these assumptions or the comparison was between a quantitative and a semi-quantitative descriptor, the Kruskal-

Wallis test, a non-parametric one-way analysis of variance, was used (Legendre &

Legendre 2012). A Dunn test (Dunn 1964) with Bonferroni correction was then used for the identification of significantly different groups. These analyses were performed with the function kruskal_test() of the package coin (Hothorn & al. 2008) with 100 000 permutations and the function dunn.test() of the package dunn.test (Dinno 2017).

4.4. Results

Soil extractions. The number of nematodes recovered from soil varied significantly between each combination of extraction methods and soil types (p <

0.0001). The highest efficiency was obtained with the Baermann funnel on muck soil with a 73.3% recovery rate while the lowest (20.5%) was also obtained in muck soil but using sugar flotation (Fig. 7). There was no significant difference between the Baermann pan and the Baermann funnel on any type of soil and the average recovery rate for these two methods was 63.8%. The efficacy of the sugar flotation method was much less with an average recovery of 24.1%. Within each method, no significant differences among soil types were found although slight variations were observed.

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.

Garlic tissue extractions. There was no significant difference between Baermann pan, Baermann funnel and the sonication method to extract stem and bulb nematodes from garlic bulbs (Table 1). However, there was a significant difference between the extraction techniques when tested on a mixture of stems and leaves. The Baermann pan and funnel methods were comparable on this kind of material and way better than the sonication method that yielded 95% less nematodes on average.

Table 1. Number of nematodes extracted from garlic bulbs or from stems and leaves using three different extraction methods. Type of garlic tissues1 Extraction methods Bulbs Stems and leaves Baermann pan 90.4 ± 19.6 ns 581.5 ± 82.1 b Baermann funnel 172.5 ± 46.0 ns 301.6 ± 80.2 b Sonication 125.4 ± 22.3 ns 22.4 ± 6.2 a 1Extractions were done on 30 g of garlic bulbs or 13 g of stems and leaves cut in small pieces from a homogenous pool of naturally infested plants. The number of nematodes is expressed by means ± standard errors of ten replicates.

4.5. Discussion

Plant parasitic nematodes represent a serious threat to agriculture as thousands of species attack nearly all crops and are responsible for probably more than 100 billion dollars of losses each year (Nicol & al. 2011). Diagnosis is challenging due to the absence of specific symptoms, the diversity and complexity of morphological characteristics and the difficulty to extract nematodes from plant or soil. Official recommendations exist for the choice of extraction method for most of the species/type of sample but surprisingly, very few actual validations have been published. For the stem

and bulb nematode, D. dipsaci, most of the scientists opt for the Baermann funnel for extraction from bulbs (e.g. Roberts & Matthews 1995; Qiao et al. 2013). This method is also recommended by the EPPO to extract D. dipsaci from seeds and plant tissues (EPPO

2013). For soil, four different methods are suggested by the EPPO and scientists’ choice is less unanimous, several choosing centrifugal flotation although no valid comparison were found. Furthermore, even if a method is better than another, the actual recovery rate is most of the time unknown or vague.

In this study, we demonstrated that the Baermann methods (pan and funnel) were clearly superior to the centrifugal sugar flotation to extract D. dipsaci from soil, yielding more than twice the number of nematodes. Soil type was also assessed and no significant differences were found although muck soil was more variable between funnel and pan than sand, loam and silty clay. In the original paper describing the method, Whitehead and Hemming (1965) also found similar numbers of nematodes (other species) between clay and sand. The recovery rate of the Baermann funnel was around 67% confirming it is a good option for soil extraction but also illustrating that a significant proportion of the nematodes in soil always go unnoticed. Still, this number is better than the 50% recovery rate obtained by Miyagawa and Lear (1970) on sandy soil using the same method.

When tested with garlic bulbs as the starting material, no significant difference was observed between the Baermann extraction methods and sonication. This confirmed that the new method proposed by Tangchitsomkid et al. (2015), using ultrasound has some potential. However, the sonication method yielded very few nematodes from leaves and stems and must therefore be discarded unless further adapted for this type of materials. Another study that compared different approaches for the extraction of D.

dipsaci from seeds of the perennial plant Brachiaria ruzizensis, a common forage grass in

Brazil, also identified the Baermann funnel as the best method (Jonsson 2015).

In our study, no significant differences were found between the Baermann funnel and the Baermann pan in any situation. Because the Baermann pan is more convenient and allow the concurrent analysis of hundreds of samples, it will be preferred for all large scale experiments. However, some limitations associated with this method were raised, including its high variability between different labs, which refrains its use for large scale quantitative surveys (Den Nijs & van den Berg 2013). For the diagnostic of D. dipsaci, this should not be problematic as any positive detection, even a very low number of nematodes, should be considered as a threat. Thus, a presence or absence result is most of the time sufficient. Even if the variability increases the chance of a false negative, this can be attenuated with appropriate sampling. Several other methods, e.g. elutriation or

Fenwick can, were not tested in this work. Some are not adapted for this species while others have shown poor results or require excessive labour time or expensive equipment.

Thus, we are confident that the Baermann pan is a valid option for the rapid evaluation of seed cloves or soil for contamination with the stem and bulb nematodes.

4.6. Acknowledgement This research was carried out with financial support from Agriculture and Agri-

Food Canada.

Connecting statement

Symptoms of plant disease like those caused by D. dipsaci could be due to several factors. Among them, many species of plant parasitic nematode could be responsible for diseases and crop losses. Detecting and identifying theses species with accuracy is therefore essential. Diagnostic of the presence of D. dipsaci, while challenging is crucial to the implantation of effective management technique. The validation of the extraction techniques confirmed the efficiency of the Baermann pan and the Baermann funnel to extract D. dipsaci from soil and plant tissues.

Although morphological identification provides information on the nematode species, with D. dipsaci, the host range, which depend on the biological race will rest unknown with this method alone. Racial characterization is then necessary to establish the host range of this pest. The purpose of the following chapter is to determine the host range of the population of D. dipsaci from eastern Canada. Combined with the genetic characterization of this pest, this project provide the knowledge necessary to evaluated the risk for other cultures while give the information to establish an efficient crop rotation plan. The extraction techniques evaluated in the previous chapter are therefore good choices for diagnostics and were used for the second and third objective of this thesis.

Chapter 5: Host range and genetic characterization of Ditylenchus dipsaci populations from Eastern Canada

5.1. Abstract

The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode affecting over 500 plant species worldwide. Since 2012, garlic producers from Ontario and Quebec have been particularly affected with economic losses caused by this pest.

Reproduction of D. dipsaci on a particular host depends on its biological race, which are unknown for these populations from Eastern Canada. As a polyphagous pest, D. dipsaci can possibly be a threat and have negative impact on many crops grown in Quebec, such as field and vegetable crops (e.g. onion). In this study, the host range of four populations of D. dipsaci from Quebec and Ontario was determined in a greenhouse experiment using

11 crops. Garlic, onion and green onion showed high susceptibility to the nematode while reproduction on potato was poor. No reproduction was observed on corn, soybean, barley, alfalfa, mustard, carrot, and lettuce. These crops could therefore be used as rotational crops in a control program. Thirty-two populations of D. dipsaci were also genetically characterized using genotyping-by-sequencing. The comparison of allele frequencies at 481 loci 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 greenhouse to preclude the possibility of a different race.

5.2. Introduction

The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode causing important economic damage worldwide on a wide variety of plants such as: garlic, onion, carrot, broad beans, alfalfa, oats, strawberry, and various ornamentals

(EPPO 2004). The species is listed as a quarantine pest by the European and

Mediterranean Plant Protection Organization (EPPO 2004). In Canada, D. dipsaci is regulated under the authority of the Plant Protection Act by the Canadian Food Inspection

Agency (CFIA) and it is considered as a quarantine pest when found on potato (CFIA

2013). Differences between countries concerning the quarantine status of D. dipsaci limit the access to the exportation market (Tenuta & al. 2014) due to the risk for important crops. This nematode has a large host range estimated at over 500 plant species separated in more than 30 different biological races (Sturhan & Brzeski 1991). For plant-parasitic nematodes, a race is composed of individuals from a population that share similar morphological characteristics but that exhibit a difference in their ability to reproduce on a set of differential hosts (Dropkin 1988). As some races of D. dipsaci are polyphagous and some host plants can sustain more than one race, racial identification of D. dipsaci is a complicated process (Janssen 1994; Viglierchio 1971). Also, because no genetic markers for racial determination is available, the only way to determine the biological race of a D. dipsaci population is to run a host range test.

The occurrence of D. dipsaci on onion and garlic was known in many provinces in Canada for a long time (Mountain 1957; Johnson & Kayler 1972; Fushtey & Kelly

1975; Hajihassani & Tenuta 2017; Government of New Brunswick 2018). However,

large outbreaks of D. dipsaci were only observed recently in garlic (Allium sativum) fields from Ontario and Quebec (RAP 2011). Large infestations can result in important crop losses of up to 90% in garlic fields (Abawi & Moktan 2010). Main symptoms on garlic and onion are swelling, distortion and malformation of leaves and bulbs. In case of severe infestation, the root basal plate can break away from the bulb, the plant turns yellow and dies and the bulb rots.

Early and effective detection of D. dipsaci is necessary for the implementation of good management practices, which are limited in the absence of host range data. Planting nematode-free garlic seeds only in non-infested sites is an essential part of a good management program against D. dipsaci (Abawi & Moktan 2010). Unfortunately, certified nematode-free garlic seeds are not currently available in Canada. Different disinfecting treatments have been proposed for contaminated seeds, including hot water bath with or without the addition of nematicides. Unfortunately, this approach is tricky as it will negatively affect seed germination and has a variable efficacy rate in killing the nematodes if the critical temperature inside the plant material is not reached (Krusberg

1961). Addition of formaldehyde was improving the method but has been abandoned due to concerns for human health (Roberts & Matthews 1995). Similarly, many soil fumigation products (e.g. methyl bromide) or nematicides that were used previously have been banned due to environmental (Solomon & Ravishankara 1992) and health hazards

(Calvert & al. 1998) Even if there are a few chemical products available, a large proportion of the garlic production in Canada is under organic management in which chemical control is not an option. Thus, crop rotation with non-host plants appears as the most sustainable (if not the only) management strategy that could be deployed to control

D. dipsaci. Finding non-host plants and determining crops at risk is therefore a priority for the management of D. dipsaci. Although essential, obtaining those data can be time- consuming as it requires greenhouse bioassays for each crop and each Ditylenchus population.

The development of genetic markers for the identification of biological race of D. dipsaci would provide an efficient tool to improve decision making in the management of this pest, but it requires a better knowledge of the genetic difference between biological races of D. dipsaci. A previous study has shown that D. dipsaci populations infesting garlic in Ontario were composed of at least two genetic groups after a cluster analysis, suggesting that there were two separate introductions of D. dipsaci in Ontario (Qiao & al.

2013). Host preference is probably dictated by many different genetic mechanisms such as the production of specific effectors by genes or receptors for plant recognition and production of molecules to bypass plant defences (Blok & al., 2008).

Thus, the elucidation of biological races in D. dipsaci will necessitate whole genome comparisons on a multitude of different populations. As whole genome sequencing of numerous samples is still expensive, genotyping-by-sequencing (GBS) (Elshire & al.

2011) represent a promising technique for that kind of study. The method has already proven to be a rapid and cost-effective method to provide genetic information and phylogenetic links in different plant-parasitic nematode species (Mimee & al. 2015).

In this study, both phenotypic and genotypic characterization was done in order to obtain better knowledge concerning the populations of D. dipsaci from Eastern Canada.

This information is essential for the development of appropriate crop rotation programs and to set the foundation for the development of molecular markers for host specificity.

5.3. Materials and methods

Nematode populations and DNA extraction. Garlic bulbs were obtained from growers and crop specialists between April 2016 and September 2017. A total of 79 samples were collected, 59 from the province of Quebec, 16 from Ontario, and 3 from

Europe. Ditylenchus sp. were extracted from 20 to 100 grams of tissues randomly picked from each sample (4 to 10 cloves) using the Baermann’s pan method (Townshend 1963).

The extraction in Baermann’s pan lasted 3-days at room temperature, instead of 24 h as suggested by the International Plant Protection Convention (2016), in order to be sure to extract all nematodes. Then, nematode samples were freeze-dried for 24 hours and DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Mississauga, ON, Canada), following the manufacturer’s instructions. Species confirmation was done using a triplex

PCR method developed by Jeszke et al. (2015) using the OneTaq® DNA polymerase kit

(Qiagen, Mississauga, ON, Canada) and the primers listed in Table 2 (Jeszke & al. 2015).

The PCR included the following steps: denaturation for 3 min at 95°C; 35 cycles at 95°C for 30 s, 62°C for 30 s and 72°C for 30 s; and a final extension at 72°C for 5 min. PCR products were separated by electrophoresis on a 2% agarose gel at 125 V during 50 min.

Table 2. Primers used in the PCR reaction to identify Ditylenchus species Species Primer ID Sequence Amplicon Reference size (bp) D. dipsaci, D. gigas, DITuniF CTGTAGGTGAACCTGC - (Jeszke & al. 2015) D. destructor D. dipsaci DITdipR GACATCACCAGTGAGCATCG 148 (Jeszke & al. 2015) D. gigas DITgigR GACCACCTGTCGATTC 270 (Jeszke & al. 2015) D. destructor DITdesR GTTTTTCGCCCACAAATTAGC 339 (Jeszke & al. 2015)

In vitro rearing. A total of four populations of D. dipsaci were reared separately in Petri dishes on sprouts of yellow pea (pea cv. Green Arrow), which is a known host for

D. dipsaci isolated from garlic in Ontario (Hajihassani & al. 2016). Two field populations of D. dipsaci, one from Quebec (QC16) and one from Ontario (ON16), were obtained from infested garlic and two D. dipsaci in-vitro cultures were obtained from the Canadian

National Collection of Insects, Arachnids and Nematodes representing the two genetic populations from Ontario described by Qiao et al. (2013).

Pea seeds were previously surface sterilised with ethanol 95% during five minutes, followed by an immersion in a 15% bleach solution during 20 minutes. Seeds were then rinsed three times (five min for each rinsing), in sterile water before being deposited on a 9-cm diam. Petri dish filled with a Nutrient agar medium (BD Difco,

Mississauga, Ontario, Canada) (one seed per Petri dish). Petri dishes were incubated during three days at room temperature, for detecting contamination from bacteria or fungus, and to allow seeds to sprout. Then, one sprouted seed was deposited on a Petri dish filled with Gamborg’s B-5 medium with minimal organics (3.2 g/L; Sigma-Aldrich), agar (8 g/L; Anachemia, CA), and sucrose (20 g/L; BioChop ®, CA).

After two weeks of growth, 15 hand-picked mixed stages juveniles and adults of

D. dipsaci of the population from Quebec (QC16) and from Ontario (ON16) were inoculated near the roots of each sprouted pea. These nematodes were previously extracted from garlic using a Baermann’s pan. Between 250 and 350 mixed stages juveniles and adults were handpicked randomly and sterilised by transferring them in contact lens solution and rinsing them in sterile water before the inoculation.

For both D. dipsaci in-vitro cultures from Ontario, nematodes were already reared on a Gamborg’s medium Petri dish. A 1-cm2 piece of Gamborg’s medium with nematodes was cut off sterile and deposited on another pea sprout in a new Gamborg’s

Petri dish. All Petri dishes were sealed with Parafilm and incubated in the dark at 23°C for at least three months.

Host range study. For this assay, 11 crops, generally cultivated in the same regions as garlic production, were tested including garlic, onion (A. cepa cv. Trail

Blazer), green onion (A. fistulosum cv. SSRBO01), soybean (Glycine max cv. P90Y90), alfalfa (Medicago sativa cv. Algonquin), barley (Hordeum vulgare cv. Polaris), corn (Zea mays cv. UT128B), potato (Solanum tuberosum cv. Chieftan), mustard (Brassica nigra cv. Caliente 119), carrot (Daucus carota subsp. sativus cv. Enterprise) and lettuce

(Lactuca sativa cv. Estival). The assay was conducted twice in the greenhouse, from

October 2016 to January 2017 and from May 2017 to August 2017, to ensure repeatability under different growing conditions. The experiment was conducted using a completely randomized factorial design with the 11 crops as the main factor and the four nematode populations (and a non-inoculated negative control) as a sub-factor with six replicates of each combination of crop and nematode population. The soil mixture was made of pasteurized beach sand (RONA, Canada) and Promix® (Premier Tech

Horticulture, Quakertown, PA, USA) in proportion of 1:3 V/V. Seeds of onion, green onion, carrot and alfalfa were planted in microcells two weeks before growing the other crops and then transplanted into 12-cm diam. polyethylene pots filled with 1.5 L of soil.

For the other crops, three to five seeds were planted in each pot. After two weeks of growth, the weakest plants were removed leaving four plants for alfalfa and barley and

only one for the other crops. Nematode inoculum for four populations of D. dipsaci, reared in vitro were prepared by wrapping the culture media into 2 Kimwipes®

(Kimberly-Clark®, Neenah, WI), immersed in tap water in glass Petri dishes, and incubated at room temperature overnight. The resulting nematode suspensions were poured through a 25-µm sieve for nematode enumeration. Nematode inoculation was done by pipetting 200 mixed stages and adult nematodes (200 µl solution of water and nematodes) on leaf axils of each plant. The inoculation was done after one month of growth for garlic, onion, carrot and alfalfa and two weeks for the other crops. Plants were grown under a 16-hour photoperiod at a day/night temperature of 22°C/20°C. Eight weeks after inoculation, aerial parts of each plant were cut, weighed and their height was measured. For some crops, like garlic, onion, green onion, carrot and potato, their bulbs or tubers were also harvested, and weighed. Nematodes were extracted from each plant

(aerial parts + bulbs or tubers when needed) using the Baermann’s pan technique and counted under a stereomicroscope (Leica Microsystems Inc., Concord, Ontario, Canada).

The number of recovered nematodes were analyzed using a Kruskal-Wallis analysis of variance performed in R (R Core Team 2017) using the kruskal_test function from the coin package (Hothorn & al. 2008) with 100 000 permutations.

Genotyping-by-sequencing. DNA extracts from 27 of the field samples as well as two reference populations from Ontario and three external D. dipsaci populations of from France and Turkey (Table 3) were used for a population genetics analysis.

Table 3. Samples of Ditylenchus dipsaci used for genetic characterization.

Country Province Region ID Canada Quebec Montérégie QC01 Canada Quebec Montérégie QC02 Canada Quebec Montérégie QC05 Canada Quebec Montérégie QC06 Canada Quebec Montérégie QC07 Canada Quebec Montérégie QC09 Canada Quebec Quebec QC10 Canada Quebec Montérégie QC12 Canada Quebec Centre-du-Quebec QC14 Canada Quebec Centre-du-Quebec QC15 Canada Quebec Montérégie QC16 Canada Quebec Montérégie QC17 Canada Quebec Laurentide QC21 Canada Quebec Chaudière-Appalaches QC24 Canada Quebec - QC25 Canada Quebec Montérégie QC27 Canada Quebec Estrie QC30 Canada Quebec Estrie QC36

Canada Quebec Estrie QC38 Canada Ontario - ON16 Canada Ontario Wellington ON42 Canada Ontario Wellington ON44 Canada Ontario Wellington ON45 Canada Ontario Wellington ON46 Canada Ontario Niagara ON47 Canada Ontario Wellington ON48 Canada Ontario Norfolk ON50 Canada Ontario Middlesex RefN Canada Ontario Hamilton RefS France - - FRP Turkey - - TUA Turkey - - TUO

DNA concentration from all samples was quantified using Qubit™ quantitation assays kit

(Thermo Fischer Scientific, Nepean, Ontario, Canada) following manufacturer’s instructions and normalized to 2 ng/µl by dilution with pure water according to the Qubit concentration for sequencing library construction.

Library preparation was done by the Genomics Analysis Core Facility at the

Institute for Integrative and Systems Biology (IBIS; Université Laval, Quebec, Canada) according to the GBS procedure developed by Elshire et al. (2011) and Mimee et al.

(2015) using a combination of Pst1/Msp1 restriction enzymes. Samples were barcoded, multiplexed and sequenced on five Ion Proton flow cells.

Processing of raw sequences and SNP calling was done using the Universal

Network Enabled Analysis Kit (UNEAK) pipeline (Lu & al. 2013) as described in

Mimee et al. (2015). This pipeline is designed to call de novo SNPs, without a reference genome with high stringency. To eliminate sequencing errors, a tolerate rate of 0.03 was used. Filtering of SNPs was done by setting the minimum call rate to 1.0 (no missing data). The minimum minor allele frequency (MAF) threshold was set to 0.01. Before analysis, SNPs were further filtered with a minimum coverage (minCov) of 20 reads and a maximum coverage (maxCov) of 10,000 reads. Clustering of D. dipsaci populations using Principal Component Analysis (PCA) was performed with the prcomp function from the stats package in R (R Core Team 2017). The fixation indexes (Fst) were calculated between each population using standard equations (Hartl & Clark 2007).

5.4. Results

Nematodes with morphological characteristics of the Ditylenchus genus were found in 45 samples, 32 from Quebec and 13 from Ontario. The molecular identification of the collected nematodes confirmed the presence of D. dipsaci in all the positive samples. An in vitro rearing was successfully initiated for two of these field populations

(one from Quebec and one from Ontario) as well as two reference populations (Ontario

South and North). All Ditylenchus populations developed very well on pea sprouts.

Nematodes were in good shape and moved well.

Host range evaluation. The host range of 11 crops was tested for four D. dipsaci populations. There were no significant differences (P > 0.05) between the host range for the two repetitions in time so the datasets were pooled before analysis. All the populations reproduced well on garlic, onion and green onion (Fig. 8). The field populations of D. dipsaci (Ontario and Quebec) showed a greater reproduction on garlic when compared to the reference populations. On the other hand, the field population from

Quebec showed a lower reproduction on green onion. Only a few nematodes were recovered from potato for all the populations. No nematode was recovered from any of the other plant tested: corn, soybean, barley, alfalfa, mustard, carrot, and lettuce.

No significant difference in plant height (P = 0.8874) or fresh weight (P =

0.7919) was observed between inoculated and non-inoculated plants.

Figure 8: Host range of 4 populations of Ditylenchus dipsaci from Quebec and Ontario. Recovered number of Ditylenchus dispaci per gram of plant tissue 8 weeks following inoculation of 4 different crop types with 200 mixed-stage juvenile nematodes/plant from four.

Genotyping-by-sequencing (GBS). The genetic diversity of 27 field populations of D. dipsaci collected from Quebec and Ontario was compared with the two reference populations from Southern and Northern Ontario as well as three international populations (France and Turkey). A total of 109,337,446 reads were used. UNEAK pipeline identified 5,617 SNPs and 481 of those were kept after filtering (MAF=0.01 and min-Cov=20) and removing loci with missing data.

PCA based on the allele frequencies at these 481 loci showed that most of the 32 populations of D. dipsaci clustered together (Fig. 9). This includes all the populations from Quebec and Ontario except one outlier sample from the Chaudière-Appalaches region in Quebec. Populations from Turkey and France were clearly separated by the first axis that explained 28.8% of the total variation.

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. Colors represent the different

origins of the populations: blue = Quebec (Canada); red = Ontario (Canada); green= Turkey; black = France. Genetic differentiation among populations of D. dipsaci was estimated by calculating the fixation index (Fst) between each pair of populations (Suppl. Table 1). The highest Fst (0.39 to 0.66) were obtained when comparing populations from France and

Turkey to Canadian populations. The outlier population from Quebec (QC24), also yielded high Fst values (0.40 to 0.56) when compared to other populations from Quebec and Ontario, supporting the result observed in the PCA. Lower Fst scores were calculated between populations from Ontario and Quebec. With the exception of the outlier QC24, the average Fst between populations from Quebec was 0.19. The lowest Fst (0.05) was observed between a population from Quebec and one from Ontario (QC17 and ON47).

This Ontarian population (ON47) showed close links (low Fst values) to all the populations from Quebec with an average Fst of 0.11 (when excluding the outlier isolate).

5.5. Discussion

The stem and bulb nematode has been known to be present in North America since the early 1930s (Hodson 1934). However, there appears to be a resurgence in recent years as major infestations are reported all over New England (Abawi & Moktan 2010),

Eastern Canada (RAP 2011), Western Canada (Hajihassani & Tenuta 2017) as well as a first mention in Ohio (Testen & al. 2014) and Minnesota (Mollov & al. 2012). As crop rotation is a critical part of an effective management plan, it is important to have a good knowledge of host plants. It is generally assumed that populations of “garlic and onion” race can develop on Allium (garlic, onion, leek, etc.), pea, snap bean and some weeds including hairy nightshade (Edwards & Taylor 1963; Sayre & Mountains 1962).

However, major variations were reported in the literature about host range. For example,

Edwards and Taylor (1963) showed that a population isolated from onion in Illinois reproduced very well on soybean and to a lesser extent on tomato. On the contrary, Sayre and Mountain (1962) tested another population, also isolated from onion around the same years in nearby southwestern Ontario that was unable to reproduce on soybean and tomato. A meta-analysis revealed that a high level of intra-racial variation exists in host range profiles of D. dipsaci, which questions the relevance of a classification system based on host specificity (Janssen 1994). Hybrids of D. dipsaci between some races were also successfully produced and exhibited host preference that differed from their parents, confirming that races are not good predictor of pathogenicity (Webster 1967).

Nevertheless, the outcome of this interbreeding confirmed that host range variation within populations is genetically controlled.

Until recently, the information about the host range of the D. dipsaci populations currently occurring in Eastern Canada was limited. A study identified yellow pea as a good host and canola as a good rotation crop for D. dipsaci (Hajihassani & al. 2016) but was limited to crops grown in the Canadian Prairies. Our results confirmed that D. dipsaci isolated from garlic in Ontario and Quebec reproduced well on garlic, green onion and onion. Production of dry onion in Quebec and Ontario represents a farm gate value of 67 million dollars (Statistics Canada 2016). Therefore, limiting the spread of this nematode becomes crucial in order to avoid important losses in garlic and in onion crops.

Although very limited, reproduction of D. dipsaci was also detected on potato. Following the precautionary principle, potato should not be grown in D. dipsaci infested fields. This recommendation is further supported by the fact that the detection of D. dipsaci in potato

fields would have important regulation repercussions because D. dipsaci is considered as a quarantine pest in Canada if found on potato plants (CFIA 2013). Fortunately, it was demonstrated that many interesting options are available as rotational crops for the management of D. dipsaci populations tested in this study, including lettuce, carrot, alfalfa, barley, corn, soybean and mustard.

Greenhouse host range evaluations are time consuming and it would be unrealistic to test every population (each field) to validate suitable rotation crops to be used at each location. Here we used a genotyping-by-sequencing approach to establish the genetic proximity of 32 populations of D. dipsaci. This technique provided thousands of SNPs with good coverage and was found to be a quick and inexpensive alternative to whole genome sequencing to highlight differences between populations. This analysis revealed that populations of D. dipsaci isolated from Quebec and Ontario were very genetically similar. The absence of difference in the host range of the four populations tested supports this finding. Also, pairwise comparisons indicated that most of the populations from Quebec had lower fixation indexes when compared to a single Ontario population

(ON47) than to their neighboring populations from Quebec. This strongly suggested that outbreak in Quebec originate from contaminated garlic cloves imported from Ontario.

Hajihassani and Tenuta (2017) reported a similar situation in Manitoba, the infestation in the Prairies originated from garlic contaminated seed pieces from Ontario.

Genetic proximity of these populations and their host range similarities obtained in this study suggest that the D. dipsaci populations from Eastern Canada could be managed with the same crop rotation program. A notable exception however is the population QC24 from the Chaudière-Appalaches region. This population was shown to

be genetically distinct and apart from the main cluster in the PCA analysis. Fixation indexes confirmed that this population was different and it is speculated that it could have been introduced many years before the large outbreak of D. dipsaci in Canada.

Phenotyping this population for crop preference will be essential for risk assessment and recommendation of crop rotation.

Demand for local garlic has exploded during the last decade in Canada. As a result, the acreage is increasing rapidly in Eastern Canada (Statistics Canada 2016).

However, the important losses associated with D. dipsaci have already convinced several growers to abandon the crop. The deployment of good management practices is therefore crucial to assure the sustainability of garlic production and to provide farmers a decent income. We have confirmed a wide selection of rotational crops to avoid D. dipsaci multiplication. Now, the development of a certification program for clean seeds will be essential to limit further contamination.

5.6. Acknowledgments

This research was carried out with financial support from Agriculture and Agri-

Food Canada. The authors would like to thank the Réseau d’Avertissements

Phytosanitaires, the Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du

Quebec (MAPAQ) and Michael Celetti for field sampling, as well as all the farmers involved in the project. The authors also thank Dr. Qing Yu from AAFC and Magali

Esquibet from INRA for D. dispaci isolates.

SUPPLMENTARY MATERIALS

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.

Chapter 6: Discussion

The stem and bulb nematode, D. dipsaci, has recently caused extensive damage to the garlic production in Eastern Canada (Celetti 2011). This pest has developed the capacity to survive harsh conditions for a long period and no pesticide is currently registered against it. Thus, management options are limited and should focus on avoiding the pest and on crop rotations (Abawi & Moktan 2010). A good diagnostic is therefore essential to prevent crop losses. However, for D. dipsaci, an accurate detection of the presence of the nematode is not sufficient as many biological races exist. Thus, knowing population phenotype is crucial for the evaluation of the risk in other crops and for the identification of plants species that could be used for crop rotation (Sturhan & Brzeski

1991).

The Baermann funnel is the most used techniques to extract plant-parasitic nematodes from soil (EPPO 2013). However, many scientists speculated that this method could be less effective with a relatively large nematode like D. dipsaci, although this was never tested. Our first hypothesis stated that the Baermann techniques (funnel and pan) were not adapted to the detection of D. dipsaci in soil and in plant samples (Chapter 4).

For soil samples, the hypothesis was that elutriation would be the most efficient method due to the recovery of both alive and death larvae (Van Bezooijen 2006). Against all odds, this technique was the less efficient one. Extraction of both larvae and soil particles with the same density make the nematodes count so hazardous that the used of this technique was abandoned for the rest of the project. As the extraction efficiency may vary depending on the type of soil (Van Bezooijen 2006), those assays were done with four

different soil textures. Extractions with the sugar flotation technique also yielded poor results, significantly lower than with the Baermann funnel or pan with all soil types.

A new method developed for migratory nematode using ultrasound

(Tangchitsomkid & al. 2015) was recently shown to be more efficient for the recovery of nematodes in plant tissues. Both garlic bulbs and garlic stems and leaves were tested.

Again, the Baermann funnel and pan showed a significantly higher recovery rate. This is probably explained by the longer soaking time when using the Baermann's techniques, which could allow the resting larvae under anhydrobiosis to rehydrated and be extracted from plant tissues. Exposing samples at longer sonication time in order to reach the same soaking time would kill the nematodes without increasing the recovery number. Based on those results, the Baermann pan, with its simple use and low cost, was recommended for

D. dipsaci diagnostic and selected to perform the other analysis of this project.

The second section of this project, explored in the chapter five, was based on the hypothesis that no difference of host range was expected between the population from

Ontario and Quebec. The host range evaluation of four populations (one from Quebec and three from Ontario) allowed the determination of crops at risk and the identification of non-host cultures for crop rotation. The worries about the other members of the Allium family like onion and green onion were confirmed; they allow a good reproduction of D. dipsaci. Furthermore, those crops represent, for Canadian growers, a much more important economic value than garlic (Statistic Canada 2016). It's then crucial to limit the impact of this pest by using good management practices. Some larvae were also found on potato. Even if their number was very limited, the presence of D. dipsaci on potato is a great concern as the pest is regulated by the CFIA on this crop. Thus, garlic and onion

growers with a confirmed D. dipsaci infestation should avoid potato in the affected area of their field. None of the four populations of D. dipsaci reproduced on lettuce, carrot, alfalfa, corn, soybean, barley and mustard. These are therefore interesting options for crop rotation. No phenotypic difference was observed between the populations from

Quebec and Ontario which is consistent with the initial hypothesis.

The third hypothesis of the project was also explored in the chapter five.

Similitudes were expected between Quebec and Ontario genotypes under the premise of a common origin. Genotyping by sequencing was found to be a good trade-off between the expensive whole genome sequencing and the less informative amplicon sequencing of few genomic regions (Mimee & al. 2015). Using, this approach, a total of 32 populations, 18 from Quebec, 11 from Ontario and 3 from Europe were compared. With the exception of one population from the region of Chaudière-Appalaches (Qc), population from Quebec and Ontario clustered in a single group indicating genetic similarities. A pairwise analysis also confirmed the low differentiation between the populations of these provinces and further supported the hypothesis of an introduction in

Quebec through a contaminated seeds lot from Ontario. Without any surprise, populations from Europe showed greater genetic distance with the Canadian isolates. This genetic distance has allowed a better evaluation of the degree of differentiation of Canadian populations and raised a red flag for one population from Quebec. The phenotype of this population from Chaudière-Appalaches will need to be evaluate to confirm its host range and limit the spread of another race if it the case. This genetic outlier probably originated from a previous introduction of D. dipsaci in Quebec.

Finally, the four populations tested in greenhouses presented the same phenotype as well as a similar genotype. It is thus possible to suggest that most of the other populations of D. dipsaci currently occurring in Eastern Canada share the same genetic background and should present the same host range characteristics. Management recommendations should therefore be the same, which simplify the choice of plants for crop rotation. However, caution is required. The presence of an outlier like the one in

Chaudière-Appalaches indicated the potential presence of another phenotypical profile of

D. dipsaci, maybe requiring other management methods. Phenotyping populations of D. dipsaci would be impossible on a large scale but should be applied for populations presenting important genetic differences. Producers and agronomists need fast answers to be able to manage this pest effectively and the genetic approached is an important tool to make better recommendation.

Chapter 7: Conclusions and contribution to knowledge

Canadian garlic growers provide a product of an exceptional quality. The culture of garlic is promising in Canada but the full potential of this crop will never be reach without an efficiency plan to control their first pest problem, the stem and bulb nematode.

The first objective of this project validated the extraction methods used with this nematode, ensuring a good diagnostic of contaminated samples. This project provided valuable information on the choice of rotational crops for D. dipsaci. It also revealed the high risks of this pest for other culture in the family of Allium such onion and green onion but also for the culture of potato. On the other hand, there is no evidence that the population currently prevailing in Eastern Canada could put the major field crops like soybean at risk. Concerning the routes of introduction and the spread of the pest, answers were brought by the genetic characterization of the eastern Canadian populations.

Through this objective, a good portrait of the situation was done and confirmed the hypothesis of an Ontarian origin for populations in Quebec. This highlighted again the need of a clean seed certification program.

More work still has to be done for a better comprehension of this pest. The whole genome sequencing of different biological races would help researchers to understand the genes implied into host specification and to develop rapid molecular diagnostic tools. The development of resistant garlic cultivars would complement the current management tools, facilitate the rotation programs and seriously hamper the reproductive and survival success of this pest in the field.

Those results revealed several urgent needs to structure the garlic industry but also highlight a very good potential to increase the importance of this crop in Canada.

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